The use of sunscreens is an established and recommended practice to protect skin from solar-induced damage. Around 30 UV filters can be used in sunscreen products in the European Union, which ought to follow the requirements of the regulation 1223/2009 to ensure their efficacy and safety for humans. Nevertheless, low photostability and putative toxicity for humans and environment have been reported for some UV filters. Particularly, the negative impact in marine organisms has recently raised concern on the scientific community. Therefore, it is important to develop new UV filters with improved safety profile and photostability. Over the last two decades, nearly 200 new compounds have revealed promising photoprotection properties. The explored compounds were obtained through different approaches, including exploration of natural sources, synthetic pathways, and nanotechnology. Almost 50 natural products and around 140 synthetic derivatives, such as benzimidazoles, benzotriazoles, hydroxycinnamic acids, xanthones, triazines, among others, have been studied aiming the discovery of novel, effective, and safer future photoprotective agents. Herein, we provide the reader with an overview about UV filters’ challenges and prospects, offering a forward-looking to the next-generation of UV filters. As mentioned before, these are some points to consider in the development of new effective and safer photoprotective agents.The stability of sunscreens is an essential requisite to ensure the photoprotection and safety. UV filters absorb UVR and enter in excited energetic levels . Then, t7)  is considered stable due to the similarity of the UV spectra before and after irradiation, as well as the minimum change of the photoprotective action, before and after the irradiation [The UV filter 3-(4-methylbenzylidene) camphor (4-MBC) (7)  suffers adiation . 11), a widely used UVA filter. The results showed the presence of around 10 photodegradation products, after 8 h of UV exposure with wavelengths of 260 or 320 nm [11 , ethylhexyl methoxycinnamate (EHMC) (21) was also studied regarding to this topic. In 1999, Tarras-Wahlberg et al. verified by high-performance liquid chromatography (HPLC) analysis that samples of 21 previously irradiated with UVR (λ = 290–320 nm) showed the presence of an additional band associated with the Z isomer of the UV filter, confirming the conversion between E and Z isomers after UV exposure , e24) was also evaluated confirming its photoinstability for UVA and photostability for UVB radiation. This compound tends to promptly decompose after irradiation, forming two photoproducts, one as a result of the loss of methyl group from the initial molecule, and the other product with an extra value of atomic number, probably associated with the oxidation of the amine group, directly linked to the aromatic ring (30) into a benzophenone derivative through a retro-aldol condensation reaction, which increases the phototoxic potential of this UV filter [The photodegradation of 2-ethylhexyl-4-dimethylaminobenzoate (Padimate O) (V filter . 11) and 2-ethylhexyl-4-dimethylaminobenzoate (24) were detected by analytic chromatographic techniques, such as gas chromatography-mass spectrometry (GC-MS) or HPLC. The number of photodegradation products varied according to the solvent used, time of exposure, and dose of radiation. The time of exposure to the radiation used ranged from 20 min to 140 h, and the solvents used for the photolysis studies reported here were water, ethyl acetate, dimethylsulfoxide (DMSO), and cyclohexane. UV filter avobenzone (11) is one of the photoprotective ingredients that shows the highest number of photodegradation products. Three photodegradation products were detected when this UV filter was exposed to the highest time of exposure (100 h) in cyclohexane [2, maintaining the solvent cyclohexane and decreasing the time of exposure to 8 h [2), but different time of exposition, type of radiation exposure and solvent, Padimate O (24) could present different photo-induced degradation products; however, all of them showed as susceptible point the aromatic amine group [In lohexane . The nume to 8 h . This phe to 8 h . Using tne group ,33. Several approaches could be used in order to ameliorate the photostability of photoprotective agents, such as the introduction of antioxidants , encapsu11) with antioxidant concentrations of 10 µg/mL, 2.5 µg/mL, and 2.5 µg/mL, respectively, as well as the improvement of the SPF value of the formulation with ubiquinone [trans-resveratrol combined with β-carotene ameliorates the photostability of the UV filters, in particular of avobenzone (11) [21) in alginate microparticles and studied its stabilisation effect in combination with vitamin E, confirming an improved capacity of stabilisation, as a result of the decrease in the photoisomerisation of the UV filter [21) photoisomerisation [10), EHMC (21), and octocrylene (30)) in hydroxypropyl-β-cyclodextrins system that successfully acted as photostabilisers [11) mediated by the combination with other UV filters, such methylbenzylidene camphor (7) or octocrylene (30) [27) [11) in order to block the conversion between keto-enol forms, and a new triazine triplet quencher molecule was added, reducing the undesirable effects of the UV filter by successful quenching the triplet excited state of UV filter [Afonso et al. (2014) reported the stabilising effect of antioxidants, namely vitamin C, vitamin E, and ubiquinone, when used concomitantly in the formulation, which improved the photostability of avobenzone (iquinone . In addione (11) . Duarte V filter . AdditioV filter ,42,43 anrisation . Al-Rawabilisers . Studiesbilisers ,46,47. Mene (30) , or with30) [27) , was pro30) [27) . For insV filter .The concern about the negative impact of UV filters on environment and organisms is growing day-by-day. The research community is trying to find alternatives and solutions to minimise the risks posed by these actual emergent pollutants . The impstratum corneum and do not penetrate into the skin [There are few studies about the toxicity for human beings. Some studies reported the ability of UV filters to penetrate the skin and reach the blood circulation, triggering concerns about the possible negative impact of UV filters in human body. Previous studies revealed that the size of the particles is a crucial parameter to consider. For instance, nanoparticles with smaller sizes could lead to a reactive response by inducing cytotoxicity . Recentlthe skin . Regardithe skin ; howeverthe skin , or mesothe skin . 2+ channel signalling and, consequently, activate certain biological processes [2+ production, and other nine activated the respective Ca2+ channel. In addition, benzylidene camphor sulfonic acid (5) and 3-benzylidene camphor (7) inhibited progesterone, and consequently induced the Ca2+ channel pathway, which leads to men’s infertility [The Organisation for Economic Co-operation and Development (OECD) published a document with standardised tests to evaluate the potential endocrine disruption molecules . In accorocesses . In totaertility . These rertility . 12) was the UV filter that tends to accumulate more among this type of compounds in placenta with concentrations between 0.25 ng/g and 5.41 ng/g [Valle-Sistac et al. (2016) also investigated the possible endocrine disrupting effects resulting from UV filters exposure in human embryos . The res30), and camphor derivatives; for instance, the double bond of the α,β-unsaturated system presents in both UV filters is known for their susceptibility to suffer Michael addition reactions with the skin proteins, forming protein adducts, that can lead to skin sensitisation reactions and to allergic contact dermatitis [Several research groups have reported other negative effects of UV filters using in vivo models, such as rats ,73, insermatitis . Sulphonrmatitis . In addirmatitis . 21) and other contaminants in freshwater fish of four Spanish rivers, with a concentration of 242 ng/g [The occurrence of UV filters in different locations worldwide with concentration values between ng/L and µg/L has been studied in lakes, seawaters, sediments, rivers, estuaries, and in aquatic organisms . In fact242 ng/g . It is n242 ng/g ,86,87. T242 ng/g . Bioaccu242 ng/g . Considezooxanthellae) that live in coral reefs depending on the coral species, the ambient, and the exposure to UVR [10) in all the coral tissues, with concentration of 31.8 ± 8.6 ng/g [10) and EHMC (21) were recently banned in sunscreens commercialised in Hawaii, due to these putative drastic consequences of coral bleaching [Acropora spp. exposed to ZnO (35) and modified particles of ZnO (35), which confirmed the bleaching caused by this specific UV filter. The modified particles (Eusolex® T2000 and Optisol™) considerably diminished the bleaching of the coral, which proved the damaging effect caused by the non-modified ZnO (35) [Since the 1990s, the effects of UVR on marine organisms started to be investigated, especially in coral reefs. Shick et al. (1996), reported some of the negative effects of UVR on coral reefs, showing the sensitivity of corals to excessive UVR exposure. These radiations also affect endosymbiotic organisms, namely dinoflagellates organisms  . Mylitus galloprovincialis [8) ; benzophenone-1  and benzophenone-3 (10) ; avobenzone (11) ; EHMC (21) (fish and mussels); octocrylene (30) ; PABA (mussels) and camphor derivatives (fish). Growth inhibition was one of the negative effects that affects algae organisms, namely Tetraselmis sp. at concentrations of 1 mg/L for homosalate (8) [Chlamydomonas reinhardtii at 5 mg/L for benzophenone-1 and benzophenone-3 (10) [C. reinhardtii [C. reinhardtii, the effect of UV filters was also evaluated in Artemia salina, a species of brine shrimp. The results were determined after 48 h of exposure and revealed that homosalate (8), avobenzone (11), and octocrylene (30) induced mortality in 54%, 64%, and 88%, respectively, at concentrations of 2 mg/L, being octocrylene (30) considered the most toxic UV filter for this organism [11) (200 µg/L) and octocrylene (30) (200 µg/L) affected the crustacean Daphia magna by reducing its ability to detect or respond to light stimuli, thus highlighting some behavioural changes [Danio renio regarding to 3-(4-methylbenzylidene) camphor (7) at concentrations between 0.19 and 0.77 mg/L [Oncorhynchus mykiss, changes in metabolic pathways, increase in leukocytes and oxidative stress were detected, using common environmental concentrations (96.0–395.6 μg/kg) of EHMC (21) [21), Padimate O (24), and octocrylene (30) were detected in Portuguese marine mussels, M. galloprovincialis and M. edulis, being octocrylene (30) detected in higher concentrations (2–7112 ng/g), in both organisms [21), PABA derivative (24), and octocrylene (30), were detected at 3992 ng/g dry weight (dw), 833 ng/g dw, and 1765 ng/g dw, respectively, in M. galloprovincialis, which evidenced the higher ability of these organisms to bioaccumulate these compounds [30) was also detected at concentration of 782 ng/g in marine mammals, specifically Pontoporia blainvillei dolphins [Bioaccumulation is the most referred effect in small marine organisms. incialis . Additiolate (8)  and greee-3 (10) . Additionhardtii . As for organism . However changes . Neuroto.77 mg/L . For fisHMC (21) . DespiteHMC (21) . EHMC  [50 = 11.75 μg/mL) and photoprotective activity exhibiting a SPF value of 22 and a maximum wavelength (λmax) of 376 nm [V. vinifera grape extracts [As for wine, olive oil by-products, such as olive leaves, also have a high content in polyphenols. Da Silva et al. (2019) studied the photoprotective and antioxidant properties of a commercial extract of ein (38) . This exf 376 nm . It is nextracts  revealedextracts , probablextracts  and the extracts . Lippia sericea [Cecropia obtusa [Acacia catechu heartwood [Lasallia pustulata [L. pustulata extract, it was verified that gyrophoric acid (39) was the major component detected by HPLC, being proposed that this secondary metabolite should be the main responsible for the antioxidant and photoprotective activities [Cecropia obtusa, it was demonstrated promising DPPH radical, superoxide radical, and singlet oxygen scavenging capacity , and the ability of maintaining the reactive oxygen species (ROS) species’ equilibrium in HaCaT cells [C. obtusa leaves extract at 20 µg/mL (SPF = 16) without cytotoxic effect in keratinocyte HaCaT cell line [A. catechu heartwood [Extracts of Brazilian  sericea , Amazonia obtusa , Acacia eartwood , Brazilieartwood , and Lasustulata  were alstivities . The othtivities ,105 and tivities  and hightivities ,105,106,aT cells . FurtheraT cells ,103,104.eartwood  could beAlpinia galanga, Curcuma longa, and Aloe vera, and proved its efficacy against photo-induced damage [Rasheed et al. (2012) developed a herbal sunscreen with extracts of d damage . Likewisd damage . Bhattacd damage . It was d damage ,112, guad damage ,114, ricd damage , and redd damage . Curdiea racovitzae and Iridaea cordata, identifying, as major constituents, three MAAs, palythine (41), asterina-330 (42), and shinorine (43), which showed antioxidant, antiaging, and photoprotective effect in HaCaT keratinocytes, being demonstrated as non-cytotoxic and non-photounstable [Sargassum cristafolium, namely, palythine (41) [Marine organisms also produce bioactive compounds with high structural complexity, due to the stress conditions in which these secondary metabolites live. Among marine natural products, MAAs are well-known by their photoprotective characteristics . There aunstable . These Mine (41) . Stigonema sp., Scytonema sp., and Lyngbya sp., was described as a potential broad-spectrum photoprotective agent due to its diverse maximum peak of absorbance  [Microcystis aeruginosa, showed photoprotective characteristics as a UV absorber, with λmax = 334 nm, due to MAAs shinorine (43) and porphyra-334 (45) [Interestingly, scytonemin (44), a pigment and secondary metabolite produced by cyanobacteria  386 nm) . Recentl 386 nm) . Other c334 (45) . Penicillium echinulatum was reported for producing photoprotective alkaloids (46–49), all the four metabolites being mainly absorbers of UVB and UVA radiation. Metabolites 48 and 49 were identified in major quantity in the collected extract, with critical wavelength (λc) of 335 nm and 334 nm, respectively [Bostrychia radicans-associated fungus Annulohypoxylon stygium presented high photostability when irradiated, showing loss of absorbance (LoA) not higher than 5% [Marine fungi ectively . Among a than 5% . However than 5% , they po than 5% .Thalassia testudinum. which demonstrated the ability to protect and repair the photoinduced UVB damage [Xiphophorus, capable of producing melanin (56), acting as an inhibitor of the formation of pyrimidine dimers, thus offering UV photoprotection [Other marine organisms were investigated for their capacity to produce photoprotective metabolites, including the marine sea grass B damage  due to totection .Among all compounds from natural extracts indicated above, catechin (36), epicatechin (37), gyrophoric acid (39), and resveratrol (40) seem to be the most promising compounds obtained from botanical extracts, considering their good photostability, photoprotective, antioxidant potential, and non-cytotoxic profile at the concentrations mentioned. Interestingly, the presence of hydroxyl groups is a common structure feature for the most promising secondary metabolites with antioxidant activity. Considering marine-derived metabolites, MAAs, palythine (41), asterina-330 (42), shinorine (43), porphyra-334 (45), and scytonemin (44) have an excellent protective ability against UVR, being 44 an UVA/UVB absorber. Comparing both botanical and marine natural sources, marine-derived extracts and metabolites possess improved ability to protect against photo-induced damage. The antioxidant potential and easily introduction in cosmetic formulations are the main strengths of botanical extracts and metabolites. Despite all the advances on analytic techniques, it is not always possible to identify the active metabolite. Moreover, the isolation and purification of the botanical and marine natural products with photoprotective activity is a time-consuming process, being obtained in a low amount, which is a major drawback for obtaining compounds to be further explored for skin care applications. Therefore, some of these natural products were used as lead compounds to obtain synthetic derivatives with promising photoprotective effects.2) was suggested as one possible UV filter. In fact, it is commercialised in some photoprotective formulations but with silica coating, due to its high photocatalytic activity, responsible for oxidation and degradation of other formulations’ components [2 was doped with Ca2+ and Zn2+ ions, which could reduce its photocatalytic activity and particle size, without interfering with its photoprotective potential [4) was reported 10 years later by Seixas and Serra (2014) [2, CePO4 was described for possessing high photocatalytic activity, low amount of white residue when applied on the skin, and increased stability [4 were evaluated, using TiO2 (34) and ZnO (35) as controls, as well as its behavioural and rheological properties, both alone and in formulation. The results revealed low interaction in formulation when combined with organic UV filters. Therefore, CePO4 is a potential future novel, stable, and efficient inorganic UV filter [Cerium oxide  . Similartability . Some paV filter .Inspired by commercialised UV filters, as well as in natural products with photoprotective properties, several compounds have been synthesised and reported as potential UV filters, with antioxidant, anti-inflammatory, and anti-photoaging activities. Herein, a reference of the novel synthetic derivatives developed with the aim of obtaining UV filtering compounds with extra pharmacological properties are presented. 30)-related compounds (57–64) were prepared and evaluated for their photoprotective effect by Polonini et al. (2014) [60, 61, and 63 displayed the best UVB protection effect, while compounds 61–63 presented the best results concerning protection against UVA. The most promising derivative was 63, which behaved as a broad band UVA/UVB filter [One of the strategies followed by research groups to obtain new organic UV filters with improved photoprotective activity is through molecular modifications of actually marketed UV filters. Eight octocrylene (. (2014) . Among tB filter . 65–68 were prepared and tested for their UV filtering properties [65 and 66 were considered as the most promising, showing photoprotective activity and non-phototoxic results, confirmed by PIF values as less than 1.3. In addition to these benzophenones, the structure-related benzophenone 69 and lactone 70 displayed UV filter properties, having lactone 70, a more potent photoprotective effect (SPF = 16), but only ability to absorb UVB radiation, contrarily to benzophenone 69, which demonstrated the ability to absorb UVA radiation [Using benzophenone derivatives as lead compounds, benzophenones operties .Compoundadiation . 71) and PABA methyl stearate (72), were prepared and evaluated for their photoprotective potential, revealing SPF values of 20.60 and 26.17, respectively [72 should avoid its penetration through the skin, making this compound a potential UV filter with a safer profile.Later, new PABA derivatives, PABA methyl ester  when compared with the control phenylbenzimidazole sulfonic acid (PBSA) (14) (96.7%) [87–89) were also prepared, being compounds with pyrrole (87), furan (88), and thiophene (89) moieties which were the most promising regarding their antioxidant, photostability, and photoprotective activities [89, followed by pyrrole derivative 87, and the furan derivative 88 [Inspired by benzimidazole and benzotriazole approved UV filters, several new heterocyclic compounds were prepared. Benzimidazole derivatives  (96.7%) . Additiotivities . Among tative 88 .90–97) were synthesised and evaluated for their photoprotective properties [90–97, 97 displayed the most promising antioxidant activity and revealed the highest SPF and UVA protection factor [Using triazine UV filters as models, new 1,3,5-triazine derivatives (operties . Among 1n factor . 7), Popiół et al. (2019) planned a small library of potential UV filters (98110) by replacing the camphor moiety by 5-arylideneimidazolidine-2,4-dione (hydantoin) while maintaining the benzylidene portion. Although the synthesised compounds revealed moderate SPF values, they demonstrated the ability to absorb both UVA and UVB radiation (λc between 339 and 391 nm) [104 and 109 were considered the less toxic against HaCaT keratinocytes and human fibroblasts cell lines, and compound 99 revealed the best UVB photoprotective properties within the tested series, with a SPF = 4.7. Some structure–activity relationships (SAR) considerations could be drawn for these derivatives. For instance, methoxy substituents at the aromatic ring are associated with photoprotection against UVA radiation; in contrast, the absence of methoxy groups in the aromatic ring is associated with interesting UVB filter properties [99, 104, and 109) and 3,4- (compounds 102 and 107) is associated with the highest values of critical wavelength, contrarily to what is observed with non-substituted benzene rings [Inspired in 3-benzylidenecamphor ( 391 nm) . Compounoperties . In addine rings .21) with ester (111–124), amide (125–126), and ketone (127) groups revealed promising UV filter activity. Interestingly, 111 and 114–127 showed multifunctional properties, combining antioxidant and photoprotective effects. Among all compounds, the derivatives 125 and 127 showed the best antioxidant activities with IC50 values lower than 8.9 ± 0.3 nmol [111 and its methylated derivative 112, aliphatic sinapate derivatives 116 and 118, and amide derivative 125 presented higher photostability than EHMC (21) [Sinapic acid analogues of EHMC (0.3 nmol . AdditioHMC (21) . 11), EHMC (21), and trans-resveratrol (40) resulted in the identification of a novel series of hybrids (128–135) with UV filter effect [max values in the range of 369 nm and 389 nm [128–135 possess antioxidant potential, with an IC50 between 88 µM and 275 µM  [128 and 131–135 possess characteristics of broad-spectrum molecules, having 128, 134, and 135 an interesting DPPH radical scavenging activity.Molecular hybridisation avobenzone (r effect . All comd 389 nm . Additioand 135) . Amongstp-hydroxycinnamic acids, and xanthones have been used as inspiration to obtain new potential UV filters. Inspired in the photoprotective activity of trans-resveratrol (40), compounds 126–141 were prepared and tested for their UV filter effect. All compounds revealed promising UV filter properties, with SPF values between 2 and 20 [Naturally occurring stilbenes, 2 and 20 .L-malate (142) is a sinapoyl ester widely described for its UV protection in plants [L-malate 142 and its analogues 143–157 were explored by Peyrot et al. (2020) [142–157, 142, 144, 147, and 150–157 showed promising photoprotective activity with LoA < avobenzone (11), and antioxidant activity, being 151 and 155–157 the most promising. Moreover, 142, 151, and 154–157 revealed photostability with LoA values less than 20% [Sinapoyl-n plants . Taking . (2020) . All comthan 20% .p-hydroxycinnamic acids, p-hydroxycinnamic diacids were prepared (158–161), being sinapic diacid (160) and caffeic diacid (161) the derivatives that displayed the best photoprotective characteristics [Based in natural-inspired eristics .162–164) with promising antioxidant activity and UV filtering characteristics [162–164 proved to absorb in the UVB range (280–320 nm). Additionally, xanthone 162 showed a dual ability to protect the skin against UV damage, through DPPH scavenging action and UV-filter capacity, without phototoxicity in the HaCaT keratinocyte cell line [165 and 166 were synthetised and evaluated for their photoprotective, antioxidant, and mutagenic activities [166 was revealed to be the most promising, displaying λc of 381 nm, confirming the ability to absorb both UVA and UVB radiations and with a SPF of 19.69 [Xanthone derivatives were studied in order to disclose their profiles as future UV filtering molecules. Resende et al. (2020) reported three hydroxylated xanthone derivatives -2-ethylhexyl-2-cyano-3-(furan-2-yl)acrylate (179) has also been described for its good capacity to absorb UVA radiation (λmax = 339 nm) and good solubility in oils for the formulation [Other potential synthetic UV filters with different scaffolds have been described, namely those with heterocyclic rings, such as the new UV absorbers PF value . -2mulation . p-hydroxycinnamic acids (180–183), furans (184–190), and pyrroles (191–193), displaying interesting UV filter properties and photostability. Moreover, p-hydroxycinnamic acid-based Meldrum’s derivatives (180 and 183) possess antioxidant and anti-tyrosinase properties, photoprotective characteristics, namely against UVA radiation and blue light, and photostability (with LoA < avobenzone (11)) [182, 184, 187, and 191, that revealed non-interaction with receptors, showing the absence of agonistic (% receptor activity < 30%) and antagonistic (% receptor activity > 70%) effects [Recently, Peyrot et al. (2020) developed a small library of compounds from Meldrum’s acid and ne (11))  reinforcne (11)) . Interes effects . 10), avobenzone (11), EHMC (21) octocrylene (30), can have in marine organisms. The decomposition of the UV filters detected in aquatic ambient was already reported, leading to the formation of toxic by-products with putative negative effects for human beings and accumulation in marine organisms. Beyond these environmental problems, UV filters can also have direct negative effects on humans, especially when photodegradation/photoisomerisation occurs. Avobenzone (11) is the UV filter most studied regarding to its photoinstability and negative effects, hence being one of the most toxic UV filters when exposed to UV radiation. The presence of certain chemical groups, such as aromatic ketones, unsaturated systems, and camphor structure, are some of the chemical moieties susceptible of inducing allergic and sensitisation skin reactions.Ultraviolet filters are incorporated in sunscreens aiming to protect the skin from the noxious effects of UV rays. Despite the strict regulation framework, new scientific evidences have raised concern about their toxic effects in humans and marine ecosystems. Neurotoxicity, endocrine disruption, malformations, decreased photosynthetic pigments, coral bleaching, and mortality, among others, are some of the confirmed negative effects that some UV filters, namely benzophenone-3 , epicatechin (37), gyrophoric acid (39), and resveratrol (40), are some of the plant-derived metabolites that could be highlighted for their photoprotective ability, but especially for their antioxidant potential due to the presence of hydroxyl groups in their structure. In addition to botanical extracts, marine secondary metabolites also exhibit photoprotection properties, namely MAAs, which are able to absorb both UVA and UVB radiation. Some derivatives inspired by marketed UV filters were also developed to overcome some of their drawbacks. From all the synthetic series presented, the camphor (7)- and avobenzone (11)-inspired derivatives, which are safer and photostable, and octocrylene (30)-inspired derivatives with improved photoprotective performance can be highlighted. Considering synthetic derivatives, benzimidazole, p-hydrocinnamic acids, quinolines, and xanthones are the base-chemical scaffolds that showed dual photoprotective–antioxidant activities. Molecular modifications are an interesting and simpler strategy to ameliorate some of characteristics of the actual commercialised UV filters. Aromatic systems, such as benzimidazole (75 and 87–89), triazine (93), xanthone (162 and 166), and quinoline (176) derivatives, are crucial for the good absorption in UV region. Additionally, the presence of other chromophores, such as the linkers that join several of these structures, with ester, amide, hydrazine groups, and double and/or triple bonds, ensure an extra effect in the final structure of the compounds, contributing to a higher ability to absorb in UVA region. Substituent groups in the aromatic scaffold, such as OH, OCH3, NH2, CN, and SO3H, among others, have been revealed to be favourable regarding the photoprotective activity, due to the presence of electronegative atoms that also allow the electronic delocalisation after irradiation. These highlighted compounds are some of the most promising in the series of the almost 150 synthetised molecules herein presented. With the aim of obtaining multifunctional compounds, the presence of at least one hydroxyl group is essential for antioxidant activity, the catechol group and 3,5-dimethoxy-4-hydroxyl pattern being the most favourable substitution moieties in the aromatic rings, which each revealed interesting antioxidant potential, namely as scavenger of the DPPH radical. Particularly p-hydroxycinnamic acid moiety and p-hydroxycinnamic diacids, compounds 151, 155–157 and 160, and 161, 180, and 183 can be highlighted as molecules with antioxidant and photoprotective activities, presenting some of them an extra ability as solubilising agents, when added to a sunscreen formulation (151 and 155–157).The existence of privileged structures in nature, produced by plants and marine organisms, is vastly known. Natural products could be directly used, after their extraction, or could inspire the creative mind of the scientists to obtain synthetic derivatives with improved efficacy and safer profile. Botanical extracts and metabolites, namely catechin (To conclude, the development of innovative, safe, effective, and non-toxic UV filters is an ongoing need and a hot research topic. Taken together, all the strategies presented in this work represent a helpful insight for the creation of the next generation UV filters with attractive and essential features.


JAK2, MPL, or CALR, with additional mutations leading to an expansion of myeloid cell lineages and, in PMF, to marrow fibrosis and cytopenias. Chronic inflammation impacting the initiation and expansion of disease in a major way has been described. Neutrophilic granulocytes play a major role in the pathogenesis of thromboembolic events via the secretion of inflammatory markers, as well as via interaction with thrombocytes and the endothelium. In this review, we discuss the molecular biology underlying myeloproliferative neoplasms and point out the central role of leukocytosis and, specifically, neutrophilic granulocytes in this group of disorders.Philadelphia chromosome negative myeloproliferative neoplasms (MPN) are composed of polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). The clinical picture is determined by constitutional symptoms and complications, including arterial and venous thromboembolic or hemorrhagic events. MPNs are characterized by mutations in  JAK2) gene, the myelproliferative leukemia virus oncogene (MPL), or the calreticulin (CALR) gene [Myeloproliferative neoplasms (MPN) form a distinct group of hematologic malignancies. According to the current WHO classification, the spectrum of MPNs include: chronic myeloid leukemia (CML); chronic neutrophilic leukemia (CNL); polycythemia vera (PV); primary myelofibrosis (PMF); essential thrombocythemia (ET); chronic eosinophilic leukemia not otherwise specified ; and otherwise unclassifiable MPNs (MPN-U) . In thisLR) gene . These tLR) gene .JAK2-positive PV and ET arguably represent the same disease entity with diagnosis determined by allelic frequency and other genetic factors, which will be described in more depth in MPNs are characterized by clonal proliferation, which will be described in JAK2V617F [JAK2 exon 12 deletion in a small fraction of patients) [MPL mutations are found in 3–10%: most prominent are the mutations MPLW515L and MPLW515K [CALR exon 9 frameshift mutations are detected in 20–25% of patients [MPNs are known to originate from a single, mutated hematopoietic stem cell (HSC), termed the MPN stem cell ,16. A haAK2V617F ,21,22,23atients)  and arouatients) ,26. In Epatients . The latpatients . In aroupatients .JAK2V617F mutations with a low allelic fraction can be found in a significant number of healthy subjects, therefore, are a common cause for clonal hematopoiesis of indeterminate potential (CHIP) [JAK2V617F mutation into an HSC resulted in myeloid-lineage biased HSC leading to erythro- and thrombocytosis in a mouse model but was also associated with increased DNA damage and low disease penetration [JAK2V617F mutations have, however, also been detected within lymphoid cell lineages, implying that malignant stem cells maintain their ability for lymphoid differentiation [JAK2V617F mutation is thought to be a driver of disease in affected patients, commonly used mouse models have not shown clonal expansion at the HSC level [JAK2V617F, the allelic burden seems to be lowest in ET and highest in MF; similarly, for CALR, the allelic burden is higher in MF than ET [ASXL1, SRSF2, and IDH2; in ET, with mutations in SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2 [ASXL-1, SRF2, U2AF1-Q157, TP53, or Ras [It is important to note that l (CHIP)  that mayl (CHIP) . Arguabll (CHIP) ,32. The etration . JAK2V61ntiation ,35. EvenSC level . TherefoSC level . As reviSC level ,39,40. ASC level ,42. The  than ET ,44,45. Sand EZH2 . Example, or Ras ,35,47.Molecular alterations on the HSC level are the underlying cause of all subtypes of MPN and can be carried across all steps of granulopoiesis, i.e., in common myeloid progenitors, granulocyte monocyte progenitors, promyelocytes, myelocytes, metamyelocytes, and, finally, mature neutrophils.JAK2V617F mutated HSCs driven by secretion of proinflammatory cytokines by stromal cells of the bone marrow [JAK2V617F mutation and assisted in the expansion of mutant clones, while having a negative effect on the non-mutant HSC [JAK2V617F-positive cell lines [JAK2V617F mutation [JAK2V617F-positive cells have been reported to be protected against JAK2 inhibition by IL-6, CXCL10, and fibroblast growth factor (FGF) [In recent years, several studies pointed out the importance of inflammation and neutrophils in the initiation and promotion of MPN ,50,51,52e marrow . For sevtant HSC . From a ll lines . In an imutation . Similaror (FGF) .JAK2 mutational status. As a mechanism, an increased binding of RUNX-1 to the NF-E2 promotor has been described in neutrophils [JAK2V617F-positive HSC in animal models [JAK2V617F allele burden, [JAK2V617F has been described in PV and ET [JAK2V617F allele burden [JAK2V617F-harboring cells, leading to a relative increase in proliferation of malignant hematopoietic stem cells compared to non-mutant cells, as the JAK2V617F mutation conferred resistance against NGAL-induced apoptosis [The transcription factor NF-E2 is often found to be overexpressed in patients with MPN , indepentrophils . Increastrophils . NF-E2 itrophils , and is trophils , as welll models . Conversl models , which il models ,65. Aparl models , as welll models . Additiol models . As summl models . Importal models , and CRP burden, ,71) leveV and ET . AlthougV and ET . YKL-40,e burden ,73,74. Ne burden ,76. Evene burden . Experimpoptosis ,78. NGALpoptosis . Table 1Leukocytosis, and especially an increase in leukocytosis, is a useful biomarker to assess risk for thrombosis and bleeding in patients’ MPN ,79,80. TIn a mouse model, thrombosis was attributed to increased neutrophilic extracellular trap (NET) formation and prevented by JAK-inhibitor treatment . By the The fact that neutrophilia in MPN is at least partially reflective of ongoing inflammation is underlined by newer gene expression profiles obtained from individuals with MPN . AlthougFor ET and PV, the prevention of thromboembolic events is a major treatment goal . EvidencJAK2 allelic burden [Interferon alpha is another cytoreductive agent which has been shown to improve constitutional symptoms and blood counts, as well as to decrease c burden ,114. Altc burden . Moreovec burden . The PROc burden . In anotc burden .Therapeutic approaches to PV (in case of failure of other available therapies) and PMF include JAK2 inhibitor treatment, most notably ruxolitinib, and, more recently, fedratinib . ImportaFedratinib has been approved in ruxolitinib-resistant MF after the phase II JAKARTA-2 trial had reported a decrease in spleen volume by one third in 55% of ruxolitinib-pretreated patients . AdditioJAK2V617F allele burden [Two novel JAK inhibitors are currently under investigation: Pancratinib (a JAK2 and FLT3 inhibitor) is effective in the reduction in spleen volume and symptoms in MF, and is especially promising in the treatment of patients with baseline cytopenias, which is usually a major limitation for treatment . It has e burden ,130.Momelotinib, apart from being an JAK1/2 inhibitor, antagonizes the effects of hepcidin; therefore, it is thought to become useful in MF with transfusion-requiring anemia. Although it decreased transfusion dependency and was non-inferior to ruxolitinib regarding spleen volume reduction, greater symptom control was achieved with ruxolitinib in a phase III trial . For ruxAlthough JAK2 inhibitors provide alleviation of symptoms, their ability to induce complete hematologic remission is limited, and complete molecular remission is not observed . AdditioJAK2, MPL, and CALR influence the phenotype of disease and how additional mutations influence the course of disease. An increase in leukocytosis, largely determined by the levels of neutrophils, is a risk factor for the development of thrombotic and hemorrhagic complications [The clinical presentation of patients with MPN depends, apart from the stage of disease and comorbidities, on the underlying molecular biology of the disease. We have discussed how mutations in ications ,79,80. Mications ,86,99. Cications . A furth


WW domain-containing oxidoreductase (WWOX) is known as one of the risk factors for Alzheimer’s disease (AD), a neurodegenerative disease. WWOX binds Tau via its C-terminal SDR domain and interacts with Tau phosphorylating enzymes ERK, JNK, and GSK-3β, and thereby limits AD progression. Loss of WWOX in newborns leads to severe neural diseases and early death. Gradual loss of WWOX protein in the hippocampus and cortex starting from middle age may slowly induce aggregation of a protein cascade that ultimately causes accumulation of extracellular amyloid beta plaques and intracellular tau tangles, along with reduction in inhibitory GABAergic interneurons, in AD patients over 70 years old. Age-related increases in pS14-WWOX accumulation in the brain promotes neuronal degeneration. Suppression of Ser14 phosphorylation by a small peptide Zfra leads to enhanced protein degradation, reduction in NF-κB-mediated inflammation, and restoration of memory loss in triple transgenic mice for AD. Intriguingly, tumor suppressors p53 and WWOX may counteract each other in vivo, which leads to upregulation of AD-related protein aggregation in the brain and lung. WWOX has numerous binding proteins. We reported that the stronger the binding between WWOX and its partners, the better the suppression of cancer growth and reduction in inflammation. In this regard, the stronger complex formation between WWOX and partners may provide a better blockade of AD progression. In this review, we describe whether and how WWOX and partner proteins control inflammatory response and protein aggregation and thereby limit AD progression. Alzheimer’s disease (AD) is the most common cause 60–75%) of dementia and an age-related neurodegenerative disease. Occurrence of AD is frequently seen in patients over 65 years old—the so called late-onset AD. For early-onset AD, the symptoms may start in patients in their 30s or 40s due to gene mutations inherited in an autosomal dominant fashion ,2,3. The% of demeExtracellular amyloid beta (Aβ) plaques around the neurons and intracellular neurofibrillary tangles (NFTs) caused by hyperphosphorylated tau proteins are considered the key markers of AD ,2,3. SigPathogenic tau, Aβ, and other proteins can be spread by transmitting from one neuron to another by exocytosis or via synapses or extracellular vesicles (EV) in the central nervous system (CNS), or transferring to the CNS from the peripheral nervous system ,11,12,13It appears that hyperphosphorylated tau protein captured in the EV is conformationally altered and is pathogenic. Tau binds and stabilizes microtubules. A small portion of intracellular tau is released to the extracellular space under physiological conditions . Tau is Presumably, tau undergoes misfolding during the period from middle to old age, and then the misfolded proteins are transmitted among neurons prior to the appearance of AD symptoms. Conceivably, misfolded tau proteins are the pathological seeds for native tau to merge and become misfolded. Phosphorylation in many specific amino acid residues in tau causes tau aggregation. Acetylated tau promotes memory loss due to disruption of the formation of spatial memories . TargetiC. elegans, no significant aggregation of amyloid β occurs [There is an age-dependent increase in protein instability and aggregation ,17,18. Tβ occurs ,5,6,7.The upstream pathway(s) that causes Aβ aggregation is largely unknown. Furthermore, the deciding factors rendering protein aggregation are not well defined. If protein aggregation starts in middle age and goes on very slowly year by year, it would be very difficult to identify and characterize the aggregation initiator(s) and the downstream protein aggregation cascade(s). Aggregated protein complexes suppress proteasomal function , and indTransforming growth factor beta (TGF-β) orchestrates the formation of extracellular matrix and controls cell growth or death. TGF-β strongly inhibits the growth of epithelial and endothelial cells ,21,22. TWe have demonstrated the presence of TGF-β-regulated protein aggregation in the hippocampi of middle-aged normal human brains. Three proteins have been identified, which are TGF-β-induced antiapoptotic factor (TIAF1), trafficking protein particle complex subunit 6A (TRAPPC6A or TPC6A), and SH3 domain-containing GRB2 such as endophilin B2 (SH3GLB2) ,28,29,30Transiently overexpressed TIAF1 polymerizes and causes degradation of membrane amyloid precursor protein (APP), followed by generation of Aβ and amyloid fibrils in vitro ,28,29,30In the following sections, we will discuss how tumor suppressor WW domain-containing oxidoreductase  limits protein aggregation ,35,36,37N-terminal WW domains containing conserved tryptophan residues, a nuclear localization signal located between the WW domains, a C-terminal short-chain alcohol dehydrogenase/reductase domain (SDR), and a proapoptotic C-terminal tail termed D3 [N-terminal first WW domain binds PPxY or PPPY motif in the target proteins [WWOX protein possesses two ermed D3 ,38,39,40ermed D3 ,35,36,37proteins ,33,39,40Second, when cells are exposed to stress stimuli, WWOX undergoes Tyr33 phosphorylation  Figure . pY33-WWN-terminal WW domain and the C-terminal SDR domain [Zfra (Zinc finger-like protein that regulates apoptosis) is a 31-amino-acid protein that binds WWOX at both the R domain . Zfra isR domain . pY33-WWR domain . The binR domain . In contR domain . The difThird, the SDR domain contains a catalytic site to bind NADPH and contributes to the redox activity of WWOX, which is involved in aerobic metabolism ,34,35,36Finally, tyrosine kinase ACK1 induces polyubiquitination of WWOX to undergo proteasomal degradation , which iA portion of WWOX is located in the cell membrane ,51,56,57Wwox deficiency leads to neurodevelopmental and degenerative neuropathies in newborns [Wwox knockout and heterozygous mice are readily subjected to seizure attack [Neuronal heterotopia or neuronal migration disorder contributes, in part, to the development of seizures ,59,60, anewborns ,62,63,64newborns . Both Wwe attack . These me attack .WWOX/Hyal-2 signaling is involved in traumatic brain injury (TBI). During TBI in rats, Hyal-2 and WWOX undergo nuclear translocation, and the Hyal-2/WWOX complex becomes accumulated in the nucleus and causes neuronal death . In vitr2+ influx in response to UV or apoptotic stress at room temperature [2+ influx. WWOXd cells undergo non-apoptotic explosion in response to UV irradiation in room temperature.In principle, WWOXf cells exhibit BCD and functional Caperature . When ceperature . Hyaluroperature ,50. HoweIn vitro studies showed that pro-survival JNK1 blocks the proapoptotic function of WWOX ,68. DuriTransiently overexpressed WWOX frequently sequesters transcription factors in the cytoplasm, and thereby blocks their transcription for prosurvival proteins in the nucleus of cancer cells in vitro ,34. HoweC-terminal SDR domain [WWOX blocks Tau hyperphosphorylation by directly binding Tau via its R domain ,69. By dR domain ,69. In cR domain ,69. TogeR domain .WWOX/Wwox gene causes severe neural diseases , metabolic disorders , and early death in newborns [WWOX-related epileptic encephalopathy (WOREE syndrome) [During mouse embryonic development, WWOX protein is highly expressed in the neural crest-derived structures such as cranial and spinal ganglia, skin pigment cells, and mesenchyme in the head, indicating the potential involvement of WWOX in neuronal differentiation and maturation ,75,76,77newborns ,73,74,75newborns . Togetheyndrome) ,71,72,73yndrome) . ExtensiWe reported that when WWOX protein is downregulated in the hippocampi of middle-aged individuals, brain protein aggregation may start to occur ,5,6,7. TWwox knockout mouse, it only takes less than 15 days after birth to let the brain proteins polymerize and aggregate in a cascade-like manner [Wwox heterozygous mice, they exhibit a faster kinetics of AD progression, compared to triple-transgenic mice [In e manner ,26,28,75Under physiological conditions, TPC6A or TPC6AΔ binds the C-terminal tail of WWOX in the cytoplasm and provides a piggyback ride for WWOX to translocate to the nucleus . The WWOWWOX prevents the aggregation of TPC6A or TPC6AΔ in the cytoplasm. Without the presence of WWOX, the fluid-phase TPC6AΔ acquires S35-phosphorylation, binds mitochondria, and undergoes polymerization. TPC6AΔ binds membrane-bound pS37-TIAF1 on the mitochondrial surface. The TPC6AΔ/TIAF1 complex stimulates activation of caspase 3, which leads to Thr688-dephosphorylation of the membrane amyloid precursor protein (APP). APP is then degraded to generate an intracellular domain (AICD) and Aβ. Aβ is secreted into the extracellular matrix to form amyloid fibrils and plaques ,6,75.When mice are subjected to traumatic brain injury or sciatic nerve dissection, endogenous WWOX is significantly upregulated and phosphorylated at Tyr33 ,42,50,80When pY33-WWOX is downregulated, pS14-WWOX is significantly increased in the lesions of AD hippocampus and cortex ,28. pS14The aforementioned pY33-pS14 transition raises the question regarding whether binding of WWOX with its protein partners contributes to the control of disease progression. We determined that Zfra4-10 or WWOX7-21 peptide strongly suppresses the growth of cancer xenografts in mice ,58. The In contrast, when Zfra4-10 and WWOX7-21 peptides are in combination in treating mice, binding of endogenous WWOX with protein partners is significantly reduced in many organs, and the cancer xenograft growth is increased .N-terminal WW domain and C-terminal SDR domain of WWOX [Finally, Zfra4-10 covalently interacts with proteins for rapid degradation in a ubiquitination/proteasome-independent manner ,81. Zfra of WWOX . Conceiv of WWOX .Tumor suppressor p53 participates in the AD progression and neurodegeneration ,83,84. Hhttps://hbatlas.org/pages/hbtd; last visit on 25 June 2021) [We selected 16 WWOX-related gene expression data from the Human Brain Transcriptome (ne 2021) ,86. Braine 2021) , no redune 2021) . No downWWOX signals with p53, Smad4, and HYAL-2 for growth suppression, apoptosis, and BCD ,36,55,76Both p53 and WWOX are guardians of the genome ,33,87,88When cells are exposed to micromolar amounts of 17-beta estradiol (E2), cell death occurs in a p53/WWOX-dependent manner . MechaniN-terminal WW domain of WWOX. Activated p53 binds TIAF1, and WWOX strengthens the p53/TIAF1 complex so as to stabilize the triad. Without p53 activation, no binding interaction occurs between TIAF1 and p53 [TIAF1 complexes with p53 and WWOX to form a triad . The TIA and p53 . Silenci and p53 . When TI and p53 , suggestWhen extracellular matrix is altered in the microenvironment, intracellular TIAF1 is upregulated and undergoes aggregation. For example, when any cells are seeded on extracellular matrices derived from other cells, these cells start to increase the intracellular levels of aggregated TIAF1 and Aβ ,27,91. TPolymerized TIAF1 binds Smad4 and blocks SMAD promoter activation . When p5While p53 and WWOX act synergistically in vitro in inducing apoptosis, it is intriguing to find that p53 may functionally antagonize with WWOX in vivo . This fuWhen in combination, p53 strongly abolishes the biological effects of WWOX, including cancer suppression and inhibition of inflammation . Most strikingly, cancer-regulated protein aggregation in the brain is blocked by WWOX . Again, Despite more than 100 years of research in AD, there are still no effective therapeutic drugs available to cure the disease ,3,7. NowIt is generally considered that there is an inverse relationship between the development of cancer and neurodegeneration in vivo ,93. It iOne of the proteins that runs in parallel in promoting cancer and neurodegeneration is pS14-WWOX ,81. pS14WWOX exhibits a plethora of functional properties in vivo. WWOX restricts cancer growth and AD progression, induces immune cell differentiation ,53,54, aUnfortunately, pS14-WWOX is significantly upregulated in the lesions of cancer  and AD hWwox heterozygous mice [Zfra4-10 and Zfra1-31 peptides are of therapeutic potential in blocking AD progression . Two potous mice . The fulous mice ,95. As sous mice ,81. SimiSecond, exogenous full-length Zfra1-31 and Zfra4-10 peptides induce memory anticancer response via expansion of Zfra-reactive spleen Z lymphocytes ,57,81. WSupporting evidence showed that the stronger the binding strength between WWOX and its partner proteins, the better the extent of cancer suppression . By the In conclusion, WWOX and its binding proteins play a critical role in limiting inflammation and associated AD progression. The stronger the binding, the better the inhibition of inflammation and suppression of AD symptoms. Presumably, when there is an elevated intracellular Zfra, Zfra binds WWOX and the Zfra/WWOX complex is subjected to degradation. Consequently, free, excessive p53 and other WWOX-binding proteins start to exert inflammatory reactions ,76.Despite WWOX being a defined risk factor for AD , functioEctopic p53 and WWOX mediate apoptosis in a synergistic manner in vitro ,53,80, wDespite several decades of global efforts, there are still no effective drugs available in curing AD. A recent monoclonal antibody-based drug Aducanumab for AD treatment has been approved by FDA . Nonethe


Amphimedon sp. via a molecular docking study, to examine their ability to act as anti-COVID-19 agents. Moreover, the pharmacokinetic properties of the most promising compounds were studied. The docking study showed that virtually screened compounds were effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and the 3CL main protease. In particular, nakinadine B (1), 20-hepacosenoic acid (11) and amphimedoside C (12) were the most promising compounds, as they demonstrated good interactions with the pockets of both enzymes. Based on the analysis of the molecular docking results, compounds 1 and 12 were selected for molecular dynamics simulation studies. Our results showed Amphimedon sp. to be a rich source for anti-COVID-19 metabolites.Since December 2019, novel coronavirus disease 2019 (COVID-19) pandemic has caused tremendous economic loss and serious health problems worldwide. In this study, we investigated 14 natural compounds isolated from  Amphimedon sp. is a rich source of potential drugs, with diverse chemical constituents exhibiting a wide range of pharmacological activities alanyl-l-valyl-n~1~--4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrrolidin-3-yl]methylbut2-enyl)-l-leucinamide) was used as a control.Most of the promising clinical trials for the treatment of COVID-19 have highlighted important antivirus drug target proteins such as 3CLpro and RdRp, which are highly conserved between SARS-CoV and 2019-nCoV, particularly in terms of functional sites. Therefore, our docking study focused on these two important targets. Recent studies have highlighted that SARS-CoV-2 genes share nucleotide identity and display 89.10% nucleotide similarity with SARS-CoV genes . The SARchain A)  was chos® coreTM  i32370 CPU @ 2.4 GHz 2.4 GHz, RAM Memory 2 GB, under the Windows 7.0 system. The docked compounds were built using Chem. 3D ultra 12.0 software ), and copied into the Discovery Studio 2.5 software suite. An automatic protein preparation module was used when applying an MMFF94 forcefield. The binding site sphere was defined automatically by the software. Next, the receptor, having been prepared as described above, was entered as the input for the “input receptor molecule” parameter in the CDOCKER protocol parameter explorer. Force fields were applied to the test compounds to obtain the lowest-energy structure. The obtained poses were studied, and those showing the best ligand–HDAC interactions were selected and used for CDOCKER energy (protein–ligand interaction energy) calculations. The receptor–ligand interactions of the complexes were examined in 2D and 3D [Docking analysis was carried out using Discovery Studio 2.5 software  and a fully automated docking tool using the “Dock ligands (CDOCKER)” protocol, running on an Intel MA, USA 10), and D and 3D ,22,23.Online property prediction was used to calculate the pharmacokinetic properties of the isolated compounds under investigation. The following properties were studied: for absorption—water solubility, Caco-2 permeability, intestinal absorption (human), skin permeability and P-glycoprotein interactions; and for distribution—VDss, Fu, Log BB and CNS permeability. In addition, metabolism and excretion were studied, and pkCSM pharmacokinetics used to predict the toxicity of the compounds, including hepatotoxicity, skin sensitization, etc. The results were analyzed by comparing the values obtained for the investigated compounds to the reference values from the pkCSM pharmacokinetics online property prediction .2 for 6lu7 and compound 3 for 6nur were used as starting points for the corresponding simulations. Molecular dynamics simulations were performed using the GROMACS 2019.25 molecular dynamics package . An AMBER99SB-ILDN  force field was used for simulations. All ligands were parameterized using ANTECHAMBER to generate parameters that are consistent with the General Amber Force Field (GAFF) . The AM1-BCC method was used to assign charges. ACPYPE  was used to prepare the ligands’ topologies into a format compatible with GROMACS. All simulations were performed in an explicit water environment, using the TIP3P model . Complexes were solvated in a dodecahedron box system and were neutralized with the addition of Na+ and Cl− ions. Steepest descent was used for minimization and Fmax was set to no greater than 1000 kJ mol−1 nm−1. Systems were equilibrated using NVT and NPT ensembles for a duration of 200 ps each. The temperature was sustained at 300 K using the V-rescale  algorithm. For the regulation of system pressure, a Parrinello–Rahman barostat  was used. The LINCS (LINear Constraint Solver) algorithm was used  to assess bond length constraints. The particle-mesh Ewald (PME) method  was used for long-range electrostatic calculations. For all simulations, the timestep was set to 2 fs. The Van der Waals cut-off distance was set to 1 nm. Binding free energies were calculated using the g_mmpbsa program , with the MM-PBSA method adapted for GROMACS. Binding energy consists of three energetic terms: potential energy in a vacuum, polar-solvation energy, and non-polar solvation energy. The SASA (solvent accessible surface area) model was used to calculate the non-polar solvation energy. For the calculations of the all-interaction energies, the last 5 ns  of the trajectories were used.The docking binding poses of compound 1–14. These 14 compounds, as shown in 1), ircinol A (2), 6-hydroxymanzamine A (3), 3,4-dihydromanzamine A, J N-oxide (4), manzamine D (5), 3,4-dihydro-6-hydroxymanzamine A (6), pyrinodemin D (7), 7-methyl-6-hexadecenoic acid (8), methyl 2-methoxyhexadecanoate (9), 11,15-icosadienoicacid (10), 20-hepacosenoic acid (11), amphimedoside C (12), keramaphidin B (13), and amphimedine (14) [The petroleum ether fraction was first chromatographed on a silica-gel column, and was then subjected to a sephadex LH-20 column and final purification on semi-preparative HPLC, yielding compounds ine (14) .12 was the best, forming seven H-bonds with Asp623 (2), Lys621 (2), Ala554, Aps452 and Arg553, and demonstrating one hydrophobic interaction with Arg553, as shown in Docking studies were carried out for all compounds, as shown in 1 has a CDOCKER energy of −45.4891 and a CDOCKER interaction energy of −51.245; the high CDOCKER energy draws particular attention to this compound. In addition, it is involved in the formation of four H-bonds, as is remdesivir. Compound 1 forms hydrogen bonds with the important amino acid residues Arg553 (2), Thr556, and Tyr455, and undergoes two pi-cation interactions with Arg624 and Asp623 and one hydrophobic interaction with Asp623, as illustrated in 11  with very similar total binding energies: −45.1, −47.9, and −44.8 kJ/mol, respectively. A comparison of the total contribution energies  of the common interacting amino acid residues of the RdRp protein for compounds 1, 12, and remdesivir is presented in Compounds 1 and 12, in contrast to remdesivir, interact with Lys621 with high positive energies, which results in a repulsive effect. Both remdesivir and compound 12 interact with Asp38, Asp 164, and Asn552 with positive energies, and with Leu41, Arg553, Pro620 with negative energies, whereas compound 1 does not interact with them. Both remdesivir and compound 1 interact with Glu167 and Ala554 with positive energies, and with Lys798 with negative energies, whereas compound 12 does not interact with these residues. Compound 1 also interacts with positive total energies with the amino acid residues Glu350, Asp390, Thr556, Asp618, Glu658, Gly678, and Thr680, and interacts with negative energies with Arg392, Met542, Val557, Ala558, Cys622, and Ser682.Remdesivir has exceptionally low binding energy values with the following amino acids of the RdRp protein: Tyr455, Arg553, Lys621, Arg624, and Lys798. Compounds 12 also interacts with Asn37, Lys51, and Cys622 with negative total energies and with Asp452 with positive energy. The following amino acid residues interact exclusively with remdesivir: Lys7, Lys438, Lys545, Lys783, Arg836 interact with negative total energies, and Asp161, Glu436, Ala448, Asp761, Glu811, Asp833, Asp845 interact with positive total energies that weaken the binding of the compound. As can be seen from 1 and 12 with other (uncommon) residues results in these compounds having similar total binding energy values to remdesivir.Compound . Interestingly, out of the fourteen virtually screened compounds, the same eight compounds which were active against the SARS-CoV-2 RdRp enzyme were also active against 3CL protease (PDB ID: 6LU7), albeit with some differences. Compound 1 was the most promising one, as it could bind well to the substrate binding pocket of 3CL protease (PDB ID: 6LU7) and showed significant inhibition and a lower docking score compared to the standard drug  and the highest CDOCKER interaction energy (−48.047) and formed six H-bonds with amino acid residues Cys145 (2), Ser144, Leu141, Asn142, and Ser46, in addition to displaying many hydrophobic interactions with His41, Met49, Cys145, and His163  and the highest CDOCKER interaction energy (−47.9309), and formed six H-bonds with Asn142, Phe140, Leu141, His163, Ser144, and Cys145, in addition to undergoing hydrophobic interactions with Asn142, His172, and Gln189, as shown in 11, four H-bonds were formed with residues Ser144 (2), Gly143, and Cys14, in addition to hydrophobic interactions with Leu27 and one important salt bridge with His163, which may reflect its lower CDOCKER energy (−55.0797) and CDOCKER interaction energy (−40.2121).Validation of the docking protocol was carried out by redocking the ligand N3 in 3CL protease (PDB ID: 6LU7). The RMSD value was less than 0.926 (less than 2), which indicates confidence in the produced docking results. The docking analysis of ligand N3 showed that it formed six H-bonds with amino acid residues Phe140, His163, Clu166 (3), and Gln189, in addition to undergoing many hydrophobic interactions with various amino acid residues, as illustrated in Figure 6ard drug . Compound His163 . Surpris10, as shown in 8, three H-bonds were formed with the residues His41, Gly143, and Cys154, in addition to two hydrophobic interactions with Met49 and Met165. Notably, compound 9 formed only two H-bonds, with the residues Cys145 and Gly143, and hydrophobic interaction occurred with Leu27 and Thr26; however, it displayed a good CDOCKER energy (−39.2038) and CDOCKER interaction energy (−39.0384), as shown in 7 formed one H-bond with the amino acid residue Gln189 and underwent one hydrophobic interaction with the residue Met49. It displayed a CDOCKER energy of −22.9961 and a CDOCKER interaction energy of −46.8449, as shown in 14, which was the least active against the SARS-CoV-2 RdRp enzyme, was also the least active against 3CL protease (PDB ID: 6LU7), with a CDOCKER energy of −4.5213 and a CDOCKER interaction energy of −37.3417, as shown in 2, 3, 4, 5, 6, and 13 showed positive CDOCKER energy, indicating that they are inactive against 3CL protease (PDB ID: 6LU7).In compound 1), 20-hepacosenoic acid (11), and amphimedoside C (12), fit well into the pockets of both enzymes and may be effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and 3Cl main protease.The docking study revealed that the virtually screened compounds, in particular the compounds nakinadine B  (PDB ID 6LU7), are relatively stable  of the interacting amino acid residues of the SARS-CoV-2 3CL protease for compounds 1 and 12 is presented in A comparison of the total contribution energies , have higher binding affinity to Met49 and Ser46. Compound 1 has exceptionally high binding affinity towards Thr45 and Glu166. Compound 12 has a higher binding affinity to Met165 than compound 1, but lower affinity compared to the reference ligand. Reference ligand N3 has a higher binding affinity to Leu27, Met165, and Pro168, in comparison to compounds 1 and 12. All three compounds interact with Gln189 with relatively similar low total binding energies. Compounds 1 (nakinadine B) and 12 (amphimedoside C) both interact with the SARS-CoV-2 RdRp and 3CL protease with low binding energies. Amphimedoside C has a much lower binding energy with 3CL main protease (−127.3 kJ/mol) than nakinadine B (−69.8 kJ/mol), and is close to reference ligand N3 in this regard (−156 kJ/mol). Both nakinadine B and amphimedoside C interact with RdRp with similar low binding energies , values similar to the result for remdesivir (−44.8 kJ/mol). Energetic analysis of nakinadine B and amphimedoside C demonstrated that both compounds have high binding affinity for SARS-CoV-2 RdRp and 3CL main protease. The data obtained can be used for further drug design and the optimization of potential dual inhibitors of SARS-CoV-2 RdRp and 3CL main protease.Compounds 1, 7–12, 14) showed good values for oral absorption. They revealed high Caco2 permeability values  and high intestinal absorption percentages , which reflects their potential for good oral absorption. Furthermore, all the tested compounds displayed relatively good skin permeability, with a logKp less than −2.5. One of the most significant parameters is a drug’s ability to cross into the brain, which can increase its efficacy, as its pharmacological action can occur centrally. It also presents certain advantages in studying the side effects. We observed from our results that some compounds, such as compound 12, showed poor distribution to the brain, and some, such as compound 14, were able to penetrate the BBB readily, whereas the remainder had a reasonable ability to penetrate the blood–brain barrier. This was based on the following protocol: molecules with an IogBB > 0.3 are considered able to readily cross the blood–brain barrier, whereas molecules with an IogBB < −1 are poorly distributed to the brain. Moreover, when CNS permeability was examined, it was found that all compounds had the ability to penetrate the CNS except for compound 14, as compounds with an IogPS > −2 are considered able to penetrate the central nervous system (CNS), whereas those with IogPS < −3 are considered unable to do so. Furthermore, the volume of distribution (VDss) was measured for all the compounds. VDss is considered low if below 0.71 L/Kg (log VDss < −0.15) and high if above 2.81 L/Kg (log VDss > −0.45). We observed that compounds 7 and 14 had high VDss values, whereas compounds 8, 10, 11 and 12 had low values, and compounds 1 and 9 had moderate values. An important detoxification enzyme, chiefly found in the liver, is Cytochrome P450 [7, 10, and 12, inhibited hERG II. Furthermore, a hepatotoxicity prediction was carried out on our tested compounds; most showed no hepatotoxic effects, with the exception of compounds 1, 12, and 14. Finally, skin sensitization is a possible side effect of dermally applied products, so our compounds were tested for their skin sensitization effects. Most compounds did not show any skin sensitization effect, with the exception of compounds 8–11.Moreover, we studied the pharmacokinetic properties of the most promising eight compounds, as shown in Amphimedon sp. showed that the virtually screened compounds might be effective against the new coronavirus via the dual inhibition of SARS-CoV-2 RdRp and 3CL main protease. In particular, the compounds nakinadine B (1), 20-hepacosenoic acid (11), and amphimedoside C (12) were the most promising, as they were well accommodated in the pockets of both enzymes. Moreover, the molecular dynamic study revealed that compounds 1 (nakinadine B) and 12 (amphimedoside C) both interacted with the SARS-CoV-2 RdRp and 3CL protease with low binding energies. Amphimedoside C had the lowest binding energy with 3CL main protease (−127.3 kJ/mol), a value which is double that of nakinadine B (−69.8 kJ/mol). Both nakinadine B and amphimedoside C interact with RdRp with similarly low binding energies . Additionally, the pharmacokinetic properties of the most promising eight compounds  showed that they all had good oral absorption values. They also revealed high Caco2 permeability  and high intestinal absorption , which reflects a good potential for oral absorption with limited toxicity. Taken together, the data from this screening study indicate the importance of Amphimedon sp. as a potential source of multitargets acting on COVID-19, and highlight its potential in the rapid discovery of drugs from natural sources.The docking study of fourteen compounds from


Dicathais orbita (D. orbita) are of interest for their anti-inflammatory properties. This study evaluates the binding mechanism and potentiality of several brominated indoles  against inflammatory mediators cyclooxygenases-1/2 (COX-1/2) using molecular docking, followed by molecular dynamics simulation, along with physicochemical, drug-likeness, pharmacokinetic (pk), and toxicokinetic (tk) properties. Molecular docking identified that these indole compounds are anchored, with the main amino acid residues, positioned in the binding pocket of the COX-1/2, required for selective inhibition. Moreover, the molecular dynamics simulation based on root mean square deviation (RMSD), radius of gyration (Rg), solvent accessible surface area (SASA), and root mean square fluctuation (RMSF) analyses showed that these natural brominated molecules transit rapidly to a progressive constant configuration during binding with COX-1/2 and seem to accomplish a consistent dynamic behavior by maintaining conformational stability and compactness. The results were comparable to the Food and Drug Administration (FDA)-approved selective COX inhibitor, aspirin. Furthermore, the free energy of binding for the compounds assessed by molecular mechanics–Poisson–Boltzmann surface area (MM–PBSA) confirmed the binding capacity of indoles towards COX-1/2, with suitable binding energy values except for the polar precursor tyrindoxyl sulfate (with COX-1). The physicochemical and drug-likeness analysis showed zero violations of Lipinski’s rule, and the compounds are predicted to have excellent pharmacokinetic profiles. These indoles are projected to be non-mutagenic and free from hepatotoxicity, with no inhibition of human ether-a-go–go gene (hERG) I inhibitors, and the oral acute toxicity LD50 in rats is predicted to be similar or lower than aspirin. Overall, this work has identified a plausible mechanism for selective COX inhibition by natural marine indoles as potential therapeutic candidates for the mitigation of inflammation.Inflammation plays an important role in different chronic diseases. Brominated indoles derived from the Australian marine mollusk  Inflammation is an essential part of the immune response system  that is Inflammation involves a complex array of functional responses in a cascade of steps , involviTwo related isoforms of COX, formed from multiple genes, have been recognized: COX-1 and COX-2 . COX-1 iVillorita cyprinoides were investigated using the in silico approach for COX inhibition [D. orbita revealed 6-bromoisatin as a potent inhibitor of nitric oxide (NO), tumor necrosis factor-alpha (TNFα), and prostaglandin in vitro [From ancient times, mollusks have been used to treat inflammatory diseases . Recentlhibition . A signihibition . Bioassain vitro . Subsequin vitro . Some rein vitro ,36,37. ND. orbita—tyrindoxyl sulfate, tyrindoleninone, 6-bromoisatin, and 6,6′dibromoindirubin  ,54. The  COX-1/2 , providiD. orbita exhibited amide pi-stacked, alkyl, pi-alkyl, types of hydrogen, hydrophobic, electrostatic, and halogen interactions with the amino acid residues in COX-1 and 2, similar to that observed in standard NSAID acetylsalicylic acid or aspirin  and tyrindoxyl sulfate (green), tyrindoleninone (blue), 6-bromoisatin (magenta), and 6, 6′-dibromoindirubin (navy blue) were calculated and plotted in a time-dependent manner along with the Apo form (black) of the COX- 1/COX-2 protein .D.orbita compounds, along with aspirin, show a stable nature, such as the Apo form of COX-1. On the other hand, in In We also concluded the Rg value analysis for both apo proteins, aspirin, and compounds  to studyThe average Rg value for the apo form of the COX-1 protein (black) was 24.62 Å a. On theHence, by revealing a lower Rg value, this analysis indicates better compactness and a healthy binding pattern for all our compounds against COX-1 and COX-2.The SASA of a protein is explored as a crucial factor in protein stability and compactness in protein folding studies . The SASThe average SASA values for apo-COX-1 (black), aspirin (red), tyrindoxyl sulfate (green), tyrindoleninone (blue), 6-bromoisatin (magenta), and 6, 6′-dibromoindirubin (navy blue) were 23,842, 23,634, 23,788, 242,67, 23,617, and 23,886 Å², respectively a. On theRoot mean square fluctuation (RMSF) values of different compounds and aspirin, along with the Apo form of COX-1/2, have been calculated at every trajectory of molecular dynamics simulation to evaluate the dynamic behavior of the complexes since it estimates the flexibility of local amino acids in the complex. In this RMSF plot , peaks dThe average RMSF values for the apo protein (black) as well as aspirin (red), tyrindoxyl sulfate (green), tyrindoleninone (blue), 6-bromoisatin (magenta), and 6,6′dibromoindirubin (navy blue) for both COX-1 and COX-2 were 1.325, 1.153, 1.147, 1.364, 1.192, and 1.249 Å and 1.056, 1.085, 1.123, 1.225, 1.127, and 1.145 Å, respectively. From the RMSF plot, it can be seen that of all the compounds, aspirin, along with Apo, have the lowest and highest root means square fluctuations at the same amino acid residue with the same position. The highest fluctuations have been observed a,b in seThe molecular mechanics–Poisson–Boltzmann surface area (MM–PBSA) method has been generally used as a reliable and efficient free energy simulation approach to calculate the binding energy of protein–ligand complexes ,75,76. TAccording to , indicating the potential for the selective inhibition of COX-2. On the other hand, in A large difference in the binding energy of tyrindoleninone, 6-bromoisatin, and 6,6′dibromoindirubin complexes was also observed compared to aspirin for COX-1/2, indicating tighter binding. Notably, a steady nature has been observed for the complexes with tyrindoleninone and 6-bromoisatin, without any significant fluctuations.D. orbita compounds, observed from molecular docking against COX-1/2.From the 100 ns molecular dynamics (MD) simulation, we can conclude that RMSD, Rg, SASA and RMSF analyses validate the binding of The RMSD analysis demonstrated that upon the binding of these brominated indoles to the COX-1/COX-2, there was no change in the stability of the proteins. RMSF, Rg, and SASA analyses also revealed a strong binding pattern for tyrindoxyl sulfate, tyrindoleninone, 6-bromoisatin, and 6,6′dibromoindirubin with COX-1/COX-2.Moreover, binding free energy analysis also revealed excellent results with tyrindoleninone, 6-bromoisatin, and 6,6′dibromoindirubin complexes towards COX-1/2 and tyrindoxyl sulfate for COX-2, showing higher binding energy values compared to the aspirin complex and representing better binding affinity and stable complex formation, consistent with the conclusion of the RMSF, Rg, and SASA analyses.D. orbita secondary metabolites, were determined through SwissADME web-based tools, as described previously by Diana et al. [The physicochemical properties, as well as drug-likeness of a et al. , and theD. orbita secondary metabolites  and topological polar surface area (TPSA), according to the range set by the SwissADME web tool [As shown in web tool . This reweb tool . Notablyweb tool . The polweb tool , which tThe oral availability of the studied indole compounds is illustrated in the bioavailability radar plots . This dep-values  revealed the availability of the brominated metabolites in the GI tract [All the brominated indoles tested here are predicted to have better absorption into the intestine than aspirin . This reGI tract .−6 cm/s (80−100% Fa) [D. orbita have high Caco-2 cell permeability except 6,6′ dibromoindirubin  permeability if they have a Papp > 10 × 10100% Fa) , equival100% Fa) . All thendirubin . Neverthndirubin . It is iD. orbita secondary metabolites acted as P-glycoprotein (P-gp) inhibitors  is one of the crucial hepatic enzymes, responsible for most of the drug metabolism . CYP2D6 M server . None ofD. orbita secondary metabolite was projected to be low in comparison with aspirin  I inhibition. Notably, oral administration of D. orbita extract containing these brominated indoles did not show evidence of major clinical toxicity during in vivo toxicity evaluation [Attrition due to clinical side effects and toxicity is a major concern in drug discovery ,141. Inttoxicity . The oraaluation , althougaluation , and theTo provide some insight into the physicochemical properties that influence the strength of COX-1 and 2 binding interactions with the brominated indoles, distance-based linear modeling was undertaken. This revealed some differences in the individual properties influencing the binding of the brominated indoles to COX-1 and 2 . COX-1 bRegression models for the predicted pharmacokinetic and toxicokinetic factors of the brominated indoles revealed no significant relationship with individual physicochemical variables for intestinal absorption, tissue permeability, clearance, or oral toxicity a. HoweveD. orbita compounds) and standard aspirin were obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov accessed 1 November 2019) in sdf format and then imported into the Maestro  molecular modeling platform. The structures were introduced into the job table, and the ligands were organized using the software Ligprep from the Schrödinger suite 2018, Maestro v11.6  [The 3D structure of the ligands  . PrimariNY, USA) . https://www.rcsb.org/ accessed 1 December 2019–20 January 2020). The active site was selected for docking experiments and processed in Maestro through the protein preparation wizard facility [The X-ray crystallographic 3D structures of COX-1 , crystallized by Sidhu et al. , and COXfacility . The subReceptor grid generation was performed by Glide (grid-based ligand docking with energetics) of Schrodinger–Maestro version 11.6 . Here, van der Waals radius scaling was set to the default scaling factor of 1.00 Å and charge cut-off of 0.25 Å. A cubic box of particular dimensions was set on the centroid of the active site residues, where they were created for the receptor. The bounding box was fixed to 10 × 10 × 10 Å to identify the dynamic binding site in the target protein.D. orbita secondary metabolites towards cyclooxygenase isoform COX-1 and COX-2 binding sites. Glide flexible ligand docking was used here for docking studies [cis/trans amide bonds. Glide XP extra precision docking was also applied, keeping all docking factors as default. No bonding restraints were provided during docking calculations. Using the Monte Carlo algorithm, ligand poses  were produced for individual input molecules, and the ligand efficiency of these molecules to the COX-1/2 enzymes was predicted using the Glide docking score. Molecular docking was employed to evaluate the affinity of the binding of  studies ,151, wit− and/or Na+ ions, where the entire solvent molecules were 92,657 with a density of 0.997 gm/cm3. To carry out the simulation, the periodic boundary requirement was incorporated, with the box size 90 × 90 × 90 Å3. The minimization of initial energy for each simulation system was conducted by the simulated annealing method via the steepest gradient approach (5000 cycles). Again, molecular dynamics simulations were performed utilizing PME methods to designate long-range electrostatic connections at a cut-off distance of 8 Å at physiological conditions  [The predictions from molecular docking studies were validated using molecular dynamics simulation using YASARA Dynamics software ; the set9% NaCl) . Multipl9% NaCl) . Molecul9% NaCl)  macro an9% NaCl)  softwareAfter the molecular dynamics simulation, MM–PBSA (molecular mechanics–Poisson–Boltzmann surface area) binding free energy calculations were done for all snapshots employing YASARA software using the following formula:Binding Energy = EpotRecept + EsolvRecept + EpotLigand + EsolvLigand—EpotComplex—EsolvComplex ,159.Here, YASARA  built-inD. orbita secondary metabolites and aspirin, were predicted using the SwissADME web tools provided by the Swiss Institute of Bioinformatics to determine their physicochemical properties [D. orbita brominated indole derivatives was performed using the QikProp module executed in the Schrödinger package [http://structure.bioc.cam.ac.uk/pkcsm, accessed on 1 November 2018). In the last few years, QikProp has been widely recognized as a useful tool for screening potential drug candidates and has proven to be an innovative tool for optimizing the pharmacokinetic profile of pharmaceutically appropriate compounds [Drug ability or drug-likeness, along with the physicochemical properties of operties . SwissADoperties , establioperties . The ADM package , along wompounds . Besidesompounds . A combiDistance-based linear models and principal coordinate analysis plots were constructed in PRIMER V7 + PERMANOVA . Euclidean distance similarity matrices were constructed on the normalized quantitative biological response variables, which were then correlated to the physicochemical predictor variables using AIC selection criteria and BEST selection procedure, with marginal tests. The models were run using 999 permutations of the data.D. orbita towards COX-1/2, with additional modeling of their physicochemical, drug-likeness, and ADMET properties. Molecular docking score, stability, and compactness within the pocket of the cyclooxygenase enzymes indicated that the D. orbita brominated compounds hold promise for the regulation of inflammation, with strong and stable binding predicted through a molecular dynamics simulation study and binding energy calculations. The physicochemical, drug-likeness properties, along with the ADMET study, predict the drug/lead-like potentiality of these natural marine indoles. According to the in silico evaluation, these brominated indole derivatives are predicted to have potential use as novel COX-inhibiting anti-inflammatory agents due to numerous interactions and beneficial properties, as observed in comparison to the standard NSAID compound aspirin. The studies will be useful for directing further in vitro, in vivo, and clinical-based evaluations and for the validation of pharmacokinetic and toxicological properties of brominated indoles from natural marine mollusk D. orbita as an anti-inflammatory agent.The investigation of the inhibition of proteins with small molecules through in silico screening strategies is of great interest and has come to play a substantial role in drug design and screening. Here, we investigated the docking and molecular dynamics simulation of brominated indoles from the natural marine mollusk


Heart failure is induced by multiple pathological mechanisms, and current therapies are ineffective against heart failure with preserved ejection fraction (HFpEF). As there are limited animal models of HFpEF, its underlying mechanisms have not yet been elucidated. Here, we employed the descending aortic constriction (DAC) technique to induce chronic pressure overload in the left ventricles of Tibetan minipigs for 12 weeks. Cardiac function, pathological and cellular changes, fibrotic signaling activation, and gene expression profiles were explored. The left ventricles developed concentric hypertrophy from weeks 4 to 6 and transition to dilation starting in week 10. Notably, the left ventricular ejection fraction was maintained at >50% in the DAC group during the 12-week period. Pathological examination, biochemical analyses, and gene profile analysis revealed evidence of inflammation, fibrosis, cell death, and myofilament dephosphorylation in the myocardium of HFpEF model animals, together with gene expression shifts promoting cardiac remodeling and downregulating metabolic pathways. Furthermore, we noted the activation of several signaling proteins that impact cardiac fibrosis and remodeling, including transforming growth factor-β/SMAD family members 2/3, type I/III/V collagens, phosphatidylinositol 3-kinase, extracellular signal-regulated kinase, matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases 1 and 2, interleukins 6 and 1β, and inhibitor of κBα/nuclear factor-κB. Our findings demonstrate that this chronic pressure overload-induced porcine HFpEF model is a powerful tool to elucidate the mechanisms of this disease and translate preclinical findings. Although survival and hospitalization rates for patients with heart failure (HF) have greatly improved over the past three decades, HF remains one of the leading causes of death worldwide. European data reported that the 12-month all-cause mortality rate for hospitalized patients with HF is approximately 24% . The EurTo explore safe and more effective strategies to treat and manage HF, numerous approaches have been used to generate animal models that mimic its phenotypes , 12. AorIn this study, we employed the aortic constriction technique to induce chronic pressure overload in porcine ventricles. The pigs developed left ventricular concentric hypertrophy, followed by left ventricle dilation with a preserved ejection fraction. Cardiac remodeling, histology, gene expression profiles, and fibrotic signaling were explored. This is the first study to use descending aortic constriction (DAC) to model HFpEF, providing a powerful tool to study this condition.n = 5 each). One group underwent sham surgery; the second underwent DAC surgery. During the experimental period, the animals were housed in an AAALAC-accredited facility at the Guangdong Laboratory Animals Monitoring Institute . At this facility, the ambient temperature and humidity were 20–26°C and 40–70%, respectively, and the light cycle was 12 h day/12 h night.All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals . The protocol was approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute . Ten Tibetan minipigs  were purchased from a licensed laboratory animal facility . Minipigs were randomly assigned to two groups (Before (week 0) and after surgery , echocardiography was performed to evaluate cardiac morphology and function. Twelve weeks after surgery, animals were euthanized, and cardiac tissues were collected for histopathological and expression analyses.The descending aorta segment was selected as the constriction site. All surgical instruments were autoclaved, and aseptic techniques were applied during the surgical procedure. The operating room was located in the AAALAC-accredited facility of the Guangdong Laboratory Animals Monitoring Institute. The minipigs were fasted overnight before surgery. Ketamine hydrochloride (6 mg/kg) and midazolam (0.5 mg/kg) were used for basal anesthesia, propofol (5 mg/kg) was used for induction anesthesia, and volatile anesthetic with isoflurane (1.5–2.5%) was used to maintain general anesthesia. The animals were placed in the right lateral position and connected to a veterinary monitor . After pre-operative skin preparation, a ~15 cm-long incision was made on the thoracic skin. Electrosurgical and blunt dissection techniques were used in combination to separate the muscle layers, and the constriction site  was exposed.After determining the constriction site, two pressure sensors were placed on the segment proximal to the heart (S1) and after the constriction site  was used to evaluate cardiac morphology and function at weeks 0, 2, 4, 6, 8, 10, and 12. Following anesthesia with ketamine hydrochloride (50 mg/kg), a transducer was placed in the left or right intercostal area to obtain both B-mode and M-mode images of the left ventricular long axis, and internal aortic dimensions. The measured indices of the M-mode images included the left ventricular dimensions at the end of systole and diastole , the ventricular septum thickness at the end of systole and diastole , the posterior wall thickness at the end of systole and diastole , and the aortic root diameter at the end of systole and diastole . The left ventricular EF and fraction shortening (FS) were analyzed using software provided by Esaote Inc.g at 4°C for 30 min. For serum biochemistry tests, an automatic biochemistry analyzer  and corresponding reagents were used. Laboratory parameters measured included creatine kinase (CK), lactate dehydrogenase (LDH), and creatinine.After overnight fasting, blood samples (5 mL) were collected from the minipigs and allowed to clot for 60 min at room temperature. Serum was separated by centrifugation at 2,150 × HE staining was used to examine tissue damage to the main organs, and Sirius red staining was used to determine cardiac fibrosis. Animals were euthanized with ketamine hydrochloride at the end of the experimental period. Heart tissues , aortic vessels, and the lungs, liver, and kidneys were collected. Tissue blocks were fixed overnight in 4% paraformaldehyde. The tissues were embedded in paraffin and sectioned to 3 μm, then sequentially treated with xylene, absolute ethanol, 95% absolute ethanol, 80% absolute ethanol, 70% absolute ethanol, and pure water for dewaxing. For HE staining, the sections were incubated in hematoxylin solution  for 10 min and then with alcohol-soluble eosin  for 25 s. For Sirius red staining to detect the myocardial collagen content, left ventricle sections were incubated in Sirius red solution for 1 h and then washed with acidified water. Following dehydration and mounting, the tissue sections were observed under a microscope .g at 4°C for 30 min, and the supernatants were collected. The ELISA was performed according to the manufacturer's instructions. Optical density was measured at 450 nm using a microplate reader .A commercial ELISA kit  was used to determine serum concentrations of cardiac troponin I (cTnI) at week 12. Blood samples from the minipigs were centrifuged at 2,500 × P-values < 0.05. Furthermore, DEGs with log2 (fold change) values >0 and <0 were considered upregulated and downregulated, respectively. Gene expression profiles are presented using volcano plots. Up- and downregulated DEGs were inputted into the web-accessible gene annotation and analysis tool Metascape (https://metascape.org) to analyze enrichment in biological processes, and Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov) to analyze Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in chronic pressure-overloaded hearts. Raw RNA-seq data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with accession number GSE167643.RNA sequencing was used to profile gene expression following DAC treatment as previously described . BrieflyTo examine myocardial apoptosis, left ventricle tissues were embedded in paraffin wax. After dewaxing and sectioning, the slices were incubated with proteinase K (20 μg/mL) at 37°C for 30 min. Sections were then washed with phosphate-buffered saline (pH 7.4) and treated with 0.5% Triton X-100 at room temperature for 5 min. A TUNEL cell apoptosis detection kit containing TdT  was used to fully cover the tissue, and samples were incubated in the dark for 60 min. The sections were then mounted with a 4′, 6-diamidino-2-phenylindole reagent containing an antifluorescence quencher  and observed under a microscope .RT-qPCR was used to determine the expression of atrial natriuretic peptide (ANP), BNP, α-smooth muscle actin (SMA), tissue inhibitor of metalloproteinase 2 (TIMP2), and matrix metalloproteinase 9 (MMP9), which are associated with HF or fibrosis. Briefly, total RNA extracted from left ventricle tissue was subjected to RT-qPCR using the primers listed in The cytosolic fraction was extracted from cardiac tissues of the sham and DAC groups to detect the expression levels of proteins involved in fibrosis, cellular proliferation, and inflammatory responses. Briefly, cytosolic proteins (40 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes . After blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with a species-appropriate secondary antibody. All primary and secondary antibodies were purchased from Cell Signaling Technology . The bands were detected with Immobilon Western Chemiluminescent HRP Substrate . Band density was analyzed using ImageJ . Primary antibodies used were as follows: anti-collagen α1 , anti-phospho-SMAD family member (SMAD)2 , anti-phospho-SMAD2/SMAD3 , anti-transforming growth factor β , anti-phospho-nuclear factor-κB p65 , anti-phospho-inhibitor of κBα , anti-phospho-extracellular signal-regulated kinase , anti-interleukin (IL)6 (cat. no. 12912), anti-IL-1β (cat. no. 12703S), anti-phospho-phosphatidylinositol 3-kinase , and anti-glyceraldehyde 3-phosphate dehydrogenase .2, and proteinase inhibitors, and then centrifuged at 12,000 × g at 4°C for 15 min. Pellets were extracted with the same ice-cold lysis buffer containing 1% Triton X-100 and centrifuged at 1,100 × g to obtain myofilaments. Phosphorylation levels of myofilaments  were measured using Pro-Q Diamond Phosphoprotein Gel Stain according to the manufacturer's instructions . Briefly, myofilaments (60 μg) were resolved by SDS-PAGE on 12% gels. After fixation with 50% methanol/10% acetic acid for 30 min twice, the gels were incubated with Pro-Q Diamond solution for 90 min. Phosphorylated bands were visualized using a Gel Doc XR+ gel documentation system . Total protein was stained with Coomassie brilliant blue , and actin bands were used as loading controls.Myofilament phosphorylation levels were analyzed to examine their responses to chronic pressure overload. Myofilaments were extracted as previously described, with modifications –21. The t-tests and a two-sided P-value with a confidence level of 95% were used to determine the differences between the groups. P < 0.05 was considered significant. The graphs showing each individual data point were presented.All experimental data are presented as means ± standard errors of the means (SEMs). Raw data of the sham and TAC groups were input into GraphPad Prism 8.0  for statistics analysis. Unpaired In this study, we developed a surgical procedure to induce an HF model in Tibetan minipigs . SeveralEchocardiography showed that the aortic roots of minipigs gradually expanded over time after surgery . The ARDChanges in the LVID displayed a V-shaped pattern over time after surgery , Table 3Pathological examination was performed on the aortas, atria, ventricles, lungs, livers, spleens, and kidneys of sham and DAC minipigs 12 weeks after surgery . In DAC Next, the ventricles, atria, and ventricular septa were examined . There wCOL1A1, COL1A2, COL3A1, COL5A2, MMP16, MMP2, Fibronectin 1, Integrin Subunit Alpha V, Transforming Growth Factor Beta Receptor 3, and TIMP1  and the fibrotic markers  were significantly higher in the DAC group than in the sham group . Sirius Upon examining alterations in myofilament phosphoproteins that regulate cardiac diastolic function, we found that the phosphorylation levels of myofilament proteins  were significantly suppressed in minipigs with HFpEF induced by chronic pressure overload compared to sham pigs . Study hBoth pathological examination and GSEA suggested that inflammatory responses were critical events in the impairment of heart cells. Secretion of cytokines upon mechanical overload is known to trigger the activation of fibroblasts. Therefore, we investigated inflammatory signaling pathways and found that DAC elevated the levels of p-IκBα, p-NFκB, IL6, and IL-1β in the minipig myocardium . These rNext, we investigated whether fibrosis-associated molecular signals were activated in HFpEF. Importantly, we observed significant increases in the levels of fibroblast-secreted TGF-β, its downstream effectors p-SMAD2 and p-SMAD2/3, and proteins associated with cellular proliferation, including p-PI3K and p-ERK, following DAC . AdditioIn this study, we successfully developed a novel HFpEF model using DAC-induced chronic pressure overload in minipigs. This model was characterized by cardiac remodeling, fibrosis, tissue damage, and impaired cellular signal transduction. Echocardiography was performed to trace the aortic and ventricular morphology and cardiac contractile function over 12 weeks, demonstrating the pattern of HFpEF disease progression. In our further investigation of the mechanisms underlying the pathological and cellular responses, the fibrotic signaling pathways in the myocardium of HFpEF were explored. Based on this, we can propose a potential regulatory mechanism underlying cardiac remodeling and functional impairment in HFpEF . BrieflyHFpEF is a heterogeneous cardiac phenotype with many patterns of cardiac remodeling. These structural phenotypes include normal cardiac tissue, concentric remodeling, concentric hypertrophy, and eccentric hypertrophy . We founRegarding the selection of constriction sites for inducing pressure overload, the aortic root, and ascending, transverse, and descending trunks have been used in small and large animal models , 18, 26.Several methods have been used to develop HFpEF pig models. Deoxycorticosterone acetate (DOCA) combined with a Western diet was used to induce hypertension and hyperlipidemia in Landrace pigs for 12 weeks , and it Metabolic KEGG pathway analysis showed that the DEGs between healthy and HFpEF myocardia were involved in citrate cycle suppression, glycolysis, amino acid biosynthesis, and pyruvate metabolism, suggesting that metabolic changes are crucial in HFpEF. Our findings are consistent with a study in a murine HFpEF model of metabolic disorder, in which researchers found that β-hydroxybutyrate was an effective treatment, as it targets the acetyl-CoA pool and mitochondrial acetylation . FurtherSMAD2/3 reduces cellular expansion  can be found at: The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute .FY, WT, and XianL designed and initiated the project. SZ, WT, XianL, XiaoL, FY, HS, XZ, JW, and HC were responsible for the laboratory experiments, data analysis, and/or animal care. WP, YZ, and PB provided critical comments during experiment design and manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.