Hypophosphatasia (HPP) is a rare genetic disease caused by inactivating variants of the ALPL gene. Few data are available on the clinical presentation in Italy and/or on Italian HPP surveys.There were 30 suspected HPP patients recruited from different Italian tertiary cares. Biological samples and related clinical, biochemical, and anamnestic data were collected and the ALPL gene sequenced. Search for large genomic deletions at the ALPL locus (1p36) was done. Phylogenetic conservation and modeling were applied to infer the effect of the variants on the protein structure.There were 21 ALPL variants and one large genomic deletion found in 20 out of 30 patients. Unexpectedly, NGS-driven differential diagnosis allowed uncovering three hidden additional HPP cases, for a total of 33 HPP subjects. Eight out of 24 coding variants were novel and classified as “pathogenic”, “likely pathogenic”, and “variants of uncertain significance”. Bioinformatic analysis confirmed that all the variants strongly destabilize the homodimer structure. There were 10 cases with low ALP and high VitB6 that resulted negative to genetic testing, whereas two positive cases have an unexpected normal ALP value. No association was evident with other biochemical/clinical parameters.We present the survey of HPP Italian patients with the highest ALPL mutation rate so far reported and confirm the complexity of a prompt recognition of the syndrome, mostly for HPP in adults. Low ALP and high VitB6 values are mandatory for the genetic screening, this latter remaining the gold standard not only to confirm the clinical diagnosis but also to make differential diagnosis, to identify carriers, to avoid likely dangerous therapy in unrecognized cases. ALPL gene (NM_000478), encoding the TNSALP , which determine a decrease up to the total loss of corresponding enzymatic activity  is a rare genetic disorder affecting bone and teeth mineralization with multisystemic manifestations involving the nervous system, musculoskeletal apparatus, and kidneys , a plethora of additional manifestations, e.g., respiratory distress, muscle hypotonia, irritability, seizures, hypercalcemia/hypercalciuria, myalgias, arthralgias, and chondro- and nephrocalcinosis, define a spectrum of different severity depicted through the six phenotypic presentations, from the lethal “n adults . Insteadn adults , 4. PPi n adults . On the n adults . With reTo date, more than 400 different deleterious  ALPL variants have been identified, scattered throughout the 1,572-bp coding sequence  with a cohort of 33 HPP patients recruited in different Italian tertiary clinical cares.The Division of Medical Genetics, Fondazione IRCCS Casa Sollievo della Sofferenza, includes a regional referral center for hereditary bone metabolism disorders. The laboratory receives requests for genetic analysis from different Italian tertiary care centers. For the current study, cases were recruited from five pediatric , three endocrinology , and four bone metabolism  referral centers for adults, two medical genetic  units. From March 2017 to August 2022, 30 index individuals were enrolled for the study after preliminary biochemistry assessment confirmed the ALP value lower than the minimum range and measured at least twice and the VitB6 level higher than the maximum range (7–18 μg/l). Exclusion criteria were the assumption of drugs affecting bone turnover  and/or diseases associated with low ALP activity/Vit B6 value .Three additional patients  underwent a next-generation sequencing (NGS) multigene panel analysis sequencing for hereditary disorders leading to early-onset osteoporosis/bone fragility and were included in the final survey after the identification of an ALPL variant: (i) subject #21 was a male newborn with fractures in the absence of trauma at 30 days of life with a suspicion of “shaken baby” syndrome; (ii) subject #22 was a boy of 2 years and 10 months, with an initial diagnosis of EDS/bone fragility; (iii) patient #23, was an adult man of 65 years, with persistent osteopenia and fractures. Unexpectedly, subjects (#21) and (#23) showed a normal (repeatedly) ALP value Table 1.Totally, 48.4% of the subjects were from South Italy, 27.2% from the Islands (Sardinia and Sicily), and 24.4% from North-Middle Italy. The sex ratio was F/M = 1.72 and median age = 51.8 +/- 19.4 years. Clinical, biochemical, and anamnestic data were collected.DNA was extracted from blood withdrawal, and the classic Sanger method was performed on 25 (75.7%) out of the 33 patients: DNA was PCR amplified, and amplicons were purified  and sequenced  for the 12 coding exons  were screened through a SureSelect gene panel  designed to capture known genes associated with calcium metabolism disorders and bone fragility, including Targeted fragments were sequenced on a MiSeq platform  with MiSeq Reagent kit V3 300-cycle flow cells. Data analysis was performed considering the frequency, impact on the encoded protein, conservation, and expression of variants using distinct tools  was calculated. Analysis was performed with R  –25.Apart from early-onset cases or the ones that were resolved through the NGS-differential diagnosis , the remaining patients were firstly admitted for clinical problems related to bone pain, myalgia, fractures, nephrolithiasis, or early-onset or post-menopausal osteoporosis screening. This biased sample selection influenced the overall clinical spectrum of the cohort; instead, around 48% of patients claimed osteoporosis/low bone mass whereas 27% showed fractures at different sites. However, it is important to highlight that the whole cohort was then enrolled only after the biochemistry showed low ALP and high VitB6 Table 2.ALPL variant. Totally, 21 cases were heterozygous and two resultant compounds were heterozygous, a finding compatible with autosomal recessive inheritance (cases #5 and #10). Of the 25 identified mutated alleles , we identified 22 different variants . Accordingly, 18 were private whereas 3  recurred in 2 apparently unrelated pedigrees/index individuals. The 21 single occurrences were distributed as follows: 16 were missense, 3 frameshift, with the presumed insertion of a premature stop codon, 1 stop loss with the presumed insertion of 11 additional residues, 1 stop gain. Eight variants out of 21 (38%) were novel since they were not reported in the official database (5) . The Franklin online genomic variant interpretation tool  found to be familial, although for others, a clinical in-depth study or segregation analysis was not available, or yet concluded at the time of this report Figure 2Due to the different assays and normal ranges used in the tertiary cares, we decided to plot the percentage deviation of the ALP value from the minimum value versus the presence/absence of the ALPL mutation. The correlation between the presence of a deleterious ALPL variant and the corresponding ALP serum value was, then, resolved in the boxplot in Other biochemical parameters  were collected, but no association was found with the presence/absence of an ALPL variant, nor with the ALP value.Several SNPs were found among positive and negative patients  as benigThe multi-alignment of the Alpl ortholog clearly showed that the novel missense variants affect residues conserved up to the amphibians, or up to the fishes, strongly supporting that they could have a deleterious effect on the protein function Figure 4FoldX computed the total energy of ALPL dimers, wild-type and mutated, as a proxy of their overall stability and the van der Waals inter-residue clashes, as energy penalization factors. The free energy calculations are reported in p.(Glu298Lys) exhibited stabilizing effects when only one monomer was mutated; however, this alteration was compensated in the double-mutated dimer; on the contrary, p. showed a destabilizing effect in the mutant monomer but a stabilizing effect with two mutations. Finally, p.(Glu235Gly) did not exhibit any structural impact on the ALPL protein.A different approach was applied to the stop loss p.(*525Argfs*11), where the additional residue fragment in the protein structure could have a putative impact on the overall folding. Comparing the stability of both the monomer and the dimer with the wild type, we observed how the mutated protein was highly unstable in its monomeric form , whereas the dimeric configuration was slightly more stable than the wild-type dimer . It has to be noted that three mutants (p. Tyr28Asp), p.(Ala179Thr), and p.(Glu235Gly)) were not part of the mutant collection found in our cohort, but they were previously studied , 28 and Asp, p., sensibly higher if compared with previous studies  , 33. SucIn our survey, 10 patients with low ALP and high VitB6 values, one also having familial hypophosphatasemia, resulted to be negative. With regard to the clinical picture of these patients, whether the phenotype could be softened or worsened by the presence of alleged pathogenic variants lying in genetic regions not covered by the screening (such as deep intronic regions or 5′ and 3′UTR) or compensatory mechanisms or the action of genetic modifiers is far from being elucidated. In this regard, we kindly point out that our NGS panel contained the whole genomic (intronic and regulatory regions) of the ALPL gene, in order to identify deep intronic splicing variants, which, however, had never been found.Interestingly, patients’ enrollment was driven by biochemistry values, regardless of the clinical spectrum that, as reported in Literature data recorded the p.(Gln207Pro) variant once in a severe autosomal recessive perinatal form in compound heterozygosity with p.(Arg71Pro) in a baby girl who died 3 h after birth (2). Patient #21 carried the same 207P variant and was affected by an autosomal dominant perinatal (30 days of life) form featured by severe fractures. However, the mother and grandmother, both carriers of the same variant, had an asymptomatic form with borderline ALPL levels. The p.(Glu88Lys) variant was found once in compound heterozygosity with the p.(Pro37Leu) in an 11-month-old boy affected by nephrocalcinosis and alterations of the fontanelle . Both ouInterestingly, 2 subjects, #6 and #9 carrying the same variant, the p.D109E, both came from Sardinia and, although they denied any apparent kinship, a possible ancestor effect cannot be excluded.About the eight novel amino acid changes, the ALPL database has two different records for residue Glu84 (#18): the first was 84Val found in a family with autosomal dominant odonto-hypophosphatasia also characterized by rickets-like signs . The secNo data can be inferred from the literature about the effect of the 267Pro (#15) or 472Arg (#2) mutants. Conversely, with regard to the Phe290 variant, Mornet et al. proved that this residue, together with Glu235, Glu291, and Asp306, coordinates the binding of the calcium atoms : it can About the stop gain p.(Tyr85*) (#19) and both the frameshift mutations, p.(Ser310Profs*28) (#17) and p.Arg391Profs*14) (#11), that, presumably, introduce a premature stop codon, the mutated proteins are likely to undergo proteasomal degradation , or, if 1Profs*14There were 22 common intronic (nr 14) and exonic (nr 8) variants identified: 17 have a MAF in gnomAD > 0.05, whereas the remaining were rarer , 41, sinp.(Arg152His) was found in three patients  with a frequency six times higher than that reported in gnomAD (MAF = 1.5%): in two cases (#7 and #19), it was identified together with a pathogenic ALPL variant. Whether 152His could actually play the role of the phenotype modifier or it is in linkage disequilibrium with an actual pathogenic variant located in genetic regions not analyzed would be a hypothesis to shed light on. Moreover, it cannot be excluded that its frequency is higher in the Italian population, although there is no evidence in public databases.To the best of our knowledge, our genetic study represents the first reporting of a so high mutation rate in an Italian HPP cohort: it follows our previous work on a smaller survey  and descWe confirm the complexity of a timely recognition of the syndrome, mostly for adult forms, due the high clinical variability In this regard, the suspicious HPP can derive from the biochemical values, low ALP, and high VitB6, and not necessarily from the clinical phenotype that can be extremely variable. The clinical diagnosis must then be confirmed by the genetic analysis that, undoubtedly, represents an essential aid to identify carriers to address to the follow-up and to make the differential diagnosis, avoiding dangerous therapies in unrecognized cases.The data presented in the study are deposited in the European Variation Archive (EVA) repository at the EMBL-EBI institute, with the following accessions: Project - PRJEB61880 and Analyses - ERZ18242459 . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Conceptualization: LC, ASS, AS, and VG; methodology: LC, DT, BA, and GN; software: TB, FPe, and MM; validation: LC, FPu, and VG; formal analysis: FPu, ASS, and AS; clinical investigation: FPu, ASS, CB, FC, SCo, RF, TF, AG, CG, EG, GG, SM, AP, LP, FPi, RR, ES, PV, SCa, MCe, MCa, and AS; resources: MCa; data curation: LC; writing original draft preparation: AS and VG; writing, review, and editing: MCa, AS, and VG. All authors contributed to the article and approved the submitted version.


Dopamine (DA) agonists, as an excellent dopamine replacement therapy for patients with early and advanced Parkinson's disease (PD), play a vital role in controlling motor and several non-motor symptoms. Besides, the application of DA agonists may delay levodopa therapy and the associated risk of motor complications. Indeed, each DA agonist has unique pharmacokinetic and pharmacodynamic characteristics and therefore has different therapeutic efficacy and safety profile. The comorbidities, significant non-motor manifestations, concomitant medications, and clinical features of PD individuals should guide the selection of a specific DA agonist to provide a more patient-tailored treatment option. Thorough knowledge of DA agonists helps clinicians better balance clinical efficacy and side effects. Therefore, this review refers to recent English-written articles on DA agonist therapy for PD patients and summarizes the latest findings on non-ergot DA agonists as well as the advantages and disadvantages of each compound to help clinicians in the selection of a specific DA agonist. In addition, novel D1/D5 partial agonists and new formulations of DA agonists are also discussed. The maiThe primary cause of dopamine neurodegeneration in PD remains unclear, but there is evidence that it may be multifactorial in terms of both etiology and pathogenesis . The com1.1Dopamine replacement with levodopa remains the most effective therapeutic option for the cardinal features of PD. Although levodopa has profound benefits for PD sufferers, the duration of its effects decreases over time, known as wearing-off. Certain features may become insufficiently responsive to levodopa, with rapid switching between time spent by PD sufferers in a state of mobility (ON-time) and immobility (OFF-time) . LevodopCurrently, treatment for movement fluctuations and LID includes a combination of controlled-release levodopa with the addition of catechol-O-methyltransferase inhibitors or monoamine oxidase B inhibitors or the use of high doses of amantadine . However1.2These issues clearly emphasize the urgent need for alternative therapeutic medication to relieve the symptoms of PD, while reducing the incidence of adverse reactions. Dopamine (DA) agonists may make up for the deficiency of levodopa. DA agonists provide a promising alternative or adjunct to levodopa therapy in PD and are related to a lower risk of motor complications and dyskinesia . Indeed,Dopamine (DA) agonists exert their effect mainly by stimulating dopamine receptors in the striatum. Dopamine receptors are widely distributed not only in the central nervous system but also in the periphery. Dopamine receptors can be divided into the D1 family (D1 and D5 receptors) and D2 family  according to their biochemical and pharmacological properties . Current1). The nigrostriatal pathway is primarily involved in the regulation of motor activity and is mediated by D1, D2 and D3 receptors [There are four dopaminergic pathways in the central nervous system . Currently, clinical use of ergot DA agonists, such as bromocriptine, cabergoline, dihydroergocryptine, and pergolide, is no longer advocated due to some serious adverse reactions, including pulmonary fibrosis, ascites, valvular heart disease, pleural effusion, and pericardial effusion -32. AlthThe five others are non-ergot compounds  that are commonly used for the treatment of PD. Rotigotine, ropinirole, and pramipexole have shown equal clinical efficacy for motor symptoms in PD both as a monotherapy and as adjunctive to levodopa . ApomorpIn this review, we mainly discuss non-ergot DA agonists, which are known to have beneficial effects for patients with early and advanced PD , 35. In 2We reviewed English-written articles published in PubMed by using the keywords, such as ‘Parkinson's disease’ and‘Dopamine agonists’ with one of the following: ‘Apomorphine’, ‘Rotigotine’, ‘Pramipexole’, ‘Ropinirole’, ‘Piribedil’, ‘Impulse Control Disorders’, ‘Dopamine Agonist Withdrawal Syndrome’, and ‘Cardiological Adverse Effects’. In most cases, papers were only selected for review when there was an established rating scale or a well-described measurement of endpoints. Systematic reviews and meta-analyses were included if they reported on DA agonist’s treatment for PD. Studies with insufficient data, duplicated publications, and drugs being tested on animals were excluded. A brief overview of the preclinical evidence of DA agonists for the treatment of PD is presented in the following section; however, a full review is beyond the scope of this article.3The beneficial effects of DA agonists on motor impairments have been demonstrated in several experimental animal PD models. Before starting clinical investigations, the above-mentioned DA agonists were all evaluated in animal models of PD to evaluate their antiparkinsonian effects, pharmacokinetic, and toxicological profiles. A brief overview of the published findings from PD animal models of DA agonists will be presented in the following section.Preclinical studies have found that apomorphine, rotigotine, pramipexole, ropinirole, and piribedil were able to reverse motor impairments in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated primates, reserpine or haloperidol treated rats, and 6-hydroxydopamine (6-OHDA) lesioned rodents -43. In aPF-06412562, PF-06649751, and PF-06669571 are novel compounds with high selectivity for D1/D5 receptors and non-catechol structure. Preclinical studies have shown that non-catechol partial D1 agonists were efficacious in preclinical models of PD -50. In a44.14.1.1Apomorphine is a broad-spectrum DA agonist that interacts with both D1 family  and D2 family  receptors . ApomorpUp to now, the primary route of administration for apomorphine in PD patients has been subcutaneous, either by intermittent subcutaneous injection or continuous infusion. Due to the adverse reactions of subcutaneous administration, several alternative administration routes have been developed, some of which are under active clinical trials. An inhaled apomorphine powder (VR040) has been developed and tested in a phase IIa study of 24 PD individuals who suffered from motor fluctuations. The study found that inhaled apomorphine was well tolerated but of limited benefit . Two larIn 2016, a phase II trial involving 19 PD patients found that a new apomorphine sublingual film can rapidly and effectively convert a patient from the OFF state to the ON state as first-line therapy. Of the 19 PD subjects, 15 PD individuals achieved a full ON state within 30 minutes . In 2020Different studies also provided sufficient evidence that apomorphine is beneficial to non-motor symptoms of PD patients -69. ApomApomorphine was well tolerated by subcutaneous administration, and only mild adverse reactions, such as yawning, drowsiness, nausea, and dizziness, were observed in clinical trials , 76. Ski4.1.2Rotigotine is a non-ergot agent with high affinity for D1, D2 and D3 receptors but low affinity for D4 and D5 receptors . A singlThe beneficial effects of rotigotine on motor symptoms of PD  and the positive effects on activities of daily living  have been demonstrated in 11 clinical trials in early PD patients using rotigotine as a single therapy -84 or inDifferent studies have demonstrated that rotigotine is effective in improving non-motor manifestations of PD. Clinical studies have shown that transdermal rotigotine therapy has a significant advantage over placebo for the cure of nocturnal sleep disorders , 99. A pRotigotine was found to be efficacious and well tolerated in PD, especially for those in the advanced stage with dysphagia. Application site reaction is the most common adverse effect of rotigotine, and other adverse reactions include nausea and drowsiness . Most of4.24.2.1Pramipexole is a selective DA agonist that primarily acts on D2/D3 receptors. Its binding capacity for the D3 receptor is 7-10 times higher than that for the D2 receptor , 110. BoPramipexole immediate release has been found to be effective for depressive symptoms of PD patients . RecentlThe safety profile did not differ between pramipexole immediate release and pramipexole sustained release, with sleep attacks, somnolence, nausea, postural hypotension, dizziness, and stomach discomfort being the most common adverse effects in early PD patients and dyskinesia in advanced PD sufferers . A case 4.2.2Ropinirole primarily stimulates D2 and D3 receptors and has little affinity for D1, adrenergic and serotonergic receptors . RopinirLimited data are available on ropinirole for non-motor symptoms of PD. A post-marketing trial involving 327 patients with early and advanced PD showed improvement in anxiety and depression symptoms after 12-14 weeks of ropinirole treatment . A post The reported adverse reactions of ropinirole include hypotension, orthostatic hypotension, dizziness, and nausea. Ropinirole has also been found to be related to sinoatrial node dysfunction in PD patients. Excessive somnolence and sleep attacks were also reported in clinical tests and should be monitored carefully , 135.4.2.3Piribedil is a selective DA agonist with a strong affinity for D2/D3 receptors. It also has a low affinity for α2-adrenergic and 5-Hydroxytryptamine (5-HT) receptors , 137. PiPiribedil has also been found to be effective for non-motor symptoms of PD patients. Piribedil is the only preparation that has been established to be efficacious for apathy and should be taken into account when apathy occurs . The eff5Although the D2/D3 agonists have been well characterized, it can be concluded from the clinical and preclinical investigations that targeted activation of the D1 receptor might be a promising therapeutic strategy for PD. In addition, targeted activation of the D1 receptor may also alleviate some of the problems related to existing therapies. The development of selective D1 agonists has been limited owing to the poor oral bioavailability, short half-life period, and significant changes in cardiovascular parameters. So far, new studies on selective D1 agonists for the treatment of PD have not been reported for more than 20 years. Recently, several new D1/D5 partial agonists have been investigated for the management of PD.5.1PF-06412562 is a moderately potent DA agonist with a highly selective affinity for D1/D5 receptors. A randomized placebo-controlled study on 38 PD patients who received PF-06412562 found that the key exploration endpoint for changes in Unified PD Rating Scale part III (UPDRS-III) score was consistent across treatment groups compared to baseline and that improvements in UPDRS-III score were clinically significant. All adverse events were well tolerated, including nausea, vomiting, and fatigue, with no significant changes in cardiovascular parameters . The res5.2PF06649751 is a novel compound with high selectivity for D1/D5 receptors and a non-catechol structure . In 20185.3PF-06669571 is a novel formulation with a non-catechol structure that primarily targets the D1 receptor and has been found to be effective in preclinical models of PD . The safIn conclusion, selective dopamine D1/D5 agonists may be a new direction for the treatment of PD. However, the efficacy, tolerance, and pharmacodynamics of the potential D1/D5 agonists need to be further investigated in multicenter studies with larger sample sizes.6Despite the advantages of DA agonists, post-marketing studies on DA agonists also emphasized the development of important long-term adverse events, including dopamine agonists withdrawal syndrome (DAWS) , 149 and6.1ICD is a complication that needs special attention in the diagnosis and treatment of PD. A total of 5% of PD sufferers have one or more clinical symptoms of ICD, and the main clinical manifestations include pathological gambling, binge eating, hypersexuality, and compulsive shopping , 152. ThIn 2014, a multicenter clinical study conducted in Spain reported that oral treatment of DA agonists was found to be strongly related to the risk of ICDs compared to transdermal rotigotine, with an incidence rate of 42% and 19%, respectively . These fThe development of ICDs in PD individuals who received the treatment of DA agonists has led clinicians to switch to piribedil, which has been rarely reported to be associated with ICDs. However, several cases of ICD have been reported in PD individuals receiving piribedil over the past few years, so caution should be taken when prescribing this drug to PD patients with a prior history of ICD . ClinicaThere has been no effective therapy for ICD. Although small clinical studies have explored the efficacy of several agents for ICD, such as atypical antipsychotics, amantadine, and selective serotonin reuptake inhibitors, there is insufficient evidence of efficacy . Clinici6.2DAWS is a severe, stereotyped neuropsychiatric syndrome associated with DA agonist withdrawal that is dose-dependent and can lead to clinically significant depression or social/occupational dysfunction . PotentiCurrently, there are no clear diagnostic criteria and effective treatment for DAWS. A multicenter observational study on 51 PD individuals suggested that patients diagnosed with moderate to severe DAWS required counseling, psychological intervention as well as the reintroduction of DA agonists previously interrupted . Strateg6.3At present, there are few studies on the relationship between the use of DA agonists and heart failure. To date, no studies have offered a definitive explanation for the link between the usage of DA agonists and an increased risk of heart failure. A population-based trial of 25459 PD patients explored the relevance between the use of DA agonists and the risk of heart failure in patients with PD. The results showed that the application of pramipexole was significantly associated with the development of heart failure, especially in the first few months of treatment and in elderly patients . A largeOther cardiological side effects relevant to the use of DA agonists include sinoatrial node dysfunction associated with ropinilole and first-degree atrioventricular conduct block related to rotigotine , 165. Ca7PD patients may have different clinical responses to a DA agonist, and it is recommended to withdraw the use of DA agonist in case of troublesome adverse reactions or the absence of benefits. DA agonist therapy should be initiated at low doses and increased until beneficial clinical effects are achieved. Elderly patients are at greater risk of adverse events during DA agonists therapy, and a risk-benefit analysis should be conducted prior to initiation of DA agonists treatment according to the presence of motor complications, cognitive dysfunctions, and comorbidities . Patient7.12).Rotigotine, ropinolole, and pramipexole have presented equal clinical efficacy for the treatment of motor symptoms in PD both as a monotherapy or as adjunctive therapy combined with levodopa . Intermi7.27.2.12 and 3).Pramipexole is beneficial to control the mental complications of PD and may be a potential antidepressant . Pramipe7.2.2Rotigotine and ropinirole can improve nocturnal dyskinesia and morning dystonia and promote sleep continuity, thereby improving subjective sleep quality , 81, 1167.2.3Apomorphine and rotigotine should be given priority to patients with drooling, dysphagia, or obvious gastrointestinal dysfunction due to their optional administration routes and the enhancement of gastrointestinal activity mediated by the D1 receptor -174. Apo7.3It is important that patients be carefully evaluated for comorbidities and concomitant medications before prescribing a specific DA agonist, as all DA agonists except pramipexole are primarily metabolized in the liver, and pramipexole is mainly excreted in urine through active renal tubular secretions , 180. ThPramipexole is especially suitable for patients treated with multiple medications to minimize the onset of drug interactions. On the contrary, pramipexole should not be applied in PD individuals with advanced kidney dysfunction, and the dose must be adjusted individually in PD sufferers with mild to moderate kidney dysfunction . The assDA agonists are effective agents for the management of motor and non-motor symptoms of PD patients. The new once-daily DA agonists and transdermal preparations made the use of DA agonists easier and increased compliance of patients and adherence to treatment. Clinical studies also showed that sustained released formulations of DA agonists were superior to immediate-release formulations in alleviating OFF time. Several studies showed the efficacy of DA agonists in improving some non-motor symptoms, especially nocturnal disturbances, apathy, and depression. However, over the past few years, there have been some clinical problems with the application of DA agonists. In addition to the common side effects, such as hypotension, dizziness, nausea, and stomach discomfort related to the application of DA agonists, ICDs and DAWS are adverse reactions that clinicians should consider when using DA agonists and know how to deal with them.In conclusion, DA agonists represent an excellent treatment option for PD patients in the early stages of the disease but also in combination with levodopa and other dopaminergic drugs for advanced PD individuals. However, not all DA agonists are exactly the same. Each of these DA agonists has a unique efficacy and safety profile because of the different pharmacokinetic and pharmacodynamic properties. It is vital for clinicians to prescribe these medications at the right timing, at the right doses, and in the right combinations with other anti-parkinsonian drugs. Individual clinical characteristics, prominent non-motor symptoms, and comorbidities should all be considered in order to optimize benefits and minimize both short-term and long-term adverse reactions. At present, the existing DA agonists have not reached the ideal state in the treatment of PD. Priority should be given to the development of a new dopaminergic compound with a rapid therapeutic effect, long duration of response, a manageable route of administration, and fewer side reactions. In the future, findings on this topic would likely help tailor the appropriate drugs for each individual patient.


The article presents the role of alpha-fetoprotein in the diagnosis and monitoring of treatment for selected genetic diseases and early childhood cancers. The authors draw attention to diagnostic pitfalls related to physiological AFP production in the first year of life, inconsistencies in laboratory tests, and result interpretation.Alpha-fetoprotein (AFP) is a protein commonly found during fetal development, but its role extends beyond birth. Throughout the first year of life, AFP levels can remain high, which can potentially mask various conditions from the neurological, metabolic, hematological, endocrine, and early childhood cancer groups. Although AFP reference values and clinical utility have been established in adults, evaluating AFP levels in children during the diagnostic process, treatment, and post-treatment surveillance is still associated with numerous diagnostic pitfalls. These challenges arise from the presence of physiologically elevated AFP levels, inconsistent data obtained from different laboratory tests, and the limited population of children with oncologic diseases that have been studied. To address these issues, it is essential to establish updated reference ranges for AFP in this specific age group. A population-based study involving a statistically representative group of patients could serve as a valuable solution for this purpose. The half-life of AFP in neonatal and infant populations has been determined to be 5.5 days at birth, 11 days between 14 and 30 days after birth, and 33 days up to 4 months of age. The rate at which AFP degrades depends on factors such as birth weight, feeding method, gestational age at birth, and the additional production of this fetal protein by the neonatal liver after delivery . HoweverThe precise definition of reference ranges and clinical utility of AFP in adults contrasts with the ambiguity surrounding its application in pediatric oncology. Studies conducted in pediatric oncology since the 1970s have employed varying reference standard ranges for AFP and lack stringent reference values for biological materials other than serum, such as cerebrospinal fluid (CSF) . SeveralDifferentiating pathological AFP concentrations in newborns poses significant challenges due to the ongoing high physiological production of AFP during the fetal period. The AFP level produced by a neoplasm may not exceed the physiological concentrations observed in infancy. Therefore, it becomes particularly challenging to exclude a neoplastic process, especially in children born prematurely and up to 4 months of age ,4.Current recommendations in pediatric oncology continue to rely on AFP reference ranges that are based on tests conducted over 40 years ago, utilizing RIA tests calibrated to internal standards. However, the diagnostic tests employed today yield results that are essentially incomparable to those obtained 20–40 years ago. Furthermore, due to the lack of studies employing modern tests on newborns, the veracity of the previously reported high physiological values for the neonatal period remains uncertain.Currently, the harmonization of diagnostic tests for AFP among laboratories continues to rely on the 1975 WHO international standard. The test results are reported in international units (IU). However, the prevailing practice is to convert these results into micrograms per liter or milliliter (µg/L or µg/mL). It is usually assumed that 1200 ng of AFP corresponds to 1000 IU .Setting reference standards for AFP in pediatric oncology is challenging due to the limited number of cases available for testing. The rarity of cancer in children makes it difficult to obtain data from a larger population, resulting in a reliance on case reports and small case series. As a consequence, the reliability of reference ranges for AFP in pediatric oncology is relatively low. It may be more practical to focus on evaluating the dynamics of changes in AFP concentrations, which tend to increase for primary lesions and gradually decrease for recurrent tumors. Additionally, assessing the half-life of AFP, which is prolonged in tumors, can provide valuable information . In pediThe condition occurs with a frequency of 1:40,000 to 1:100,000 live births, making it the second most common autosomal recessive ataxia in children, following Friedrich ataxia. The underlying cause is a mutation in the ATM gene located on chromosome 11 at locus q22–23, which is responsible for DNA repair and regulation of the cell cycle by controlling the synthesis of the suppressor protein TP53. In 90% of patients, AFP levels are elevated, which distinguishes it from Friedrich ataxia ,8. This The condition occurs with a frequency of 1:1,000,000 births and follows an autosomal dominant inheritance pattern. It is associated with a de novo mutation in the ZBTB20 gene, which leads to a microdeletion at locus 3q13.31. The ZBTB20 gene acts as a key repressor of DNA transcription during birth and is responsible for various processes such as neurogenesis, fetal liver development, cell growth, detoxification, and glucose metabolism. The clinical presentation of the condition includes intellectual disability, macrocephaly, high postnatal growth, cataracts, deafness, auricular calcifications, and myopathy. The high levels of AFP observed in this condition are a consequence of the mutation in the ZBTB20 gene, which leads to the unblocking of AFP synthesis in the liver  [The condition is found in approximately 1:100,000 births and follows an autosomal recessive inheritance pattern due to a mutation in the FAH gene located on chromosome 15q23–q25. This mutation results in a deficiency of fumarylacetoacetase hydrolase, leading to the accumulation of toxic tyrosine metabolites, namely fumarylacetoacetate and maleylacetoacetate, in the liver and kidneys. These metabolites have mutagenic properties and inhibit porphobilinogen synthesis, leading to porphyria-like seizures .In the early form of the disease (<2 months of age), there is acute liver failure, which carries a high mortality rate. High levels of AFP, a marker of early liver regeneration that begins in fetal life, are particularly observed in this form. In the late form (occurring after 6 months of age), cirrhosis, hypophosphatemic rickets, and liver failure are typical. With time, the risk of liver cancer increases; it occurs in 37% of patients over 2 years of age, typically between 4 and 5 years of age. The most common form is hepatocellular carcinoma (HCC), but hepatoblastoma (HB), as well as mixed types, can also occur. AFP serves as an early marker of hepatic remodeling, neoplastic transformation, and porphyria seizures in this condition ,16,17. AThe condition occurs with a frequency of approximately 1:50,000–1:100,000 births and follows an autosomal recessive inheritance pattern. The underlying mutation affects the ABCB11 gene, located on chromosome 2 (2q24), which is responsible for encoding the BSEP protein. BSEP is a membrane transport protein found on the surface of hepatocytes. The mutation disrupts the normal transport of bile from hepatocytes to the bile ducts, resulting in its accumulation within hepatocytes. This leads to chronic inflammation and carcinogenesis. Patients with this condition typically present with jaundice and increased pruritus either in the newborn period or early childhood. It accounts for approximately 10–15% of cases of neonatal cholestasis. The disease progresses rapidly, resulting in cirrhosis, HCC (5–15% of children with PFIC-2), biliary tract cancer, and liver transplantation. AFP is a marker of increased disease progression to neoplasia ,22,23.NICCD is an autosomal recessive disorder resulting from a mutation in the SLC25A13 gene, which is responsible for encoding the citrin protein. Citrin is a mitochondrial transport protein involved in the urea cycle. A deficiency of citrin leads to serum hyperammonemia as well as abnormalities in glycolysis and beta-oxidation of fatty acids. As a result, hepatocytes are unable to utilize glucose and fatty acids as an energy source, leading to hyperlipidemia and hepatic steatosis. The condition is most commonly reported in East Asian countries such as China, Japan, and Korea, but cases have been documented worldwide. Neonatal cholestasis, accompanied by low birth weight and elevated AFP levels, is a characteristic feature. Despite these symptoms, NICCD is generally considered a benign disease, as it tends to spontaneously resolve within the first year of life with the introduction of lactose-free and/or medium-chain fatty acid (LF/MCT) nutrition. However, if left untreated, it can progress to cirrhosis, necessitating liver transplantation ,25,26.The deficiency of transaldolase, caused by a mutation in the transaldolase gene located at 11p15.5–p15, is an autosomal recessive disorder that occurs with a frequency of 1:1,000,000 births. Transaldolase is an enzyme produced in the liver that plays a role in the pentose phosphate pathway. Deficiency of this enzyme results in a defect in the pentose phosphate pathway and the accumulation of polyols in the blood, urine, and CSF. Elevated AFP levels serve as a marker of liver regeneration and tumorigenesis . In a stThe infection is primarily transmitted vertically. Approximately 90% of newborns from HbsAg and HbeAg-positive mothers will develop chronic infections if they do not receive postnatal immunoprophylaxis. In contrast to adults, where only 5–10% develop chronic hepatitis when infected, 25–90% of infected newborns experience chronic hepatitis. To predict the occurrence of HCC, determining the levels of AFP is useful. Elevated AFP levels typically coincide with increased aminotransferases and the presence of fibrosis, indicating necro-inflammatory changes. Elevated AFP levels can also be observed in asymptomatic chronic carriers with normal aminotransferase values. It is important to consider the possibility of HBV infection in patients with other tumors that secrete AFP to avoid unnecessary surgical interventions and chemotherapy . A smallThe condition occurs with a frequency of 1–5/1,000,000 births and is inherited in an autosomal recessive manner. The genetic basis of the condition involves numerous mutations, approximately 19 in total, that affect genes responsible for DNA repair. A diagnostic feature is the instability of chromosome structure following exposure to alkylating drugs. Aslan et al., based on serial measurements of serum AFP levels in pregnant carriers, demonstrated that AFP cannot serve as a marker for amniotic fluid in prenatal diagnosis, as even pregnant women carrying affected fetuses exhibit typical serum AFP levels . In thisThe level of AFP during fetal life is influenced by the levels of thyroid hormones. This is because thyroid-stimulating hormone (TSH) binds with AFP in the fetal blood plasma. It is believed that triiodothyronine (T3) plays a role in the transcriptional switch from AFP to albumin early in life. In the absence of T3 (hypothyroidism), this physiological process is delayed . T3 has In the field of pediatric oncology, AFP is utilized as a diagnostic tool and for monitoring the effectiveness of surgical treatment and chemotherapy in cases of embryonal HB, HCC, and germ cell tumors (GCTs) .HB is the most common malignant liver tumor in children, accounting for 67–80% of cases and occurring at a rate of 1–10/1,000,000 births. It represents approximately 1–2% of pediatric cancers . The tumHCC is the second most common malignant liver tumor in children, accounting for 2–33% of cases. It occurs at a rate of 0.41/1,000,000 births. Several predisposing factors contribute to its development, including the vertical transmission of HBV, tyrosinemia, progressive familial intrahepatic cholestasis, glycogen storage diseases, Alagille syndrome, congenital portal-systemic shunts, Wilson disease, alpha-1-antitrypsin deficiency, transaldolase deficiency, Gardner’s syndrome, FA, ataxia-telangiectasia, familial adenomatous polyposis, and primary sclerosing cholangitis  see Tab. HCC prip = 0.45) [GCTs account for 3.5% of cancers in children up to the age of 15 and 13.9% in the 15–19 age group . They ar = 0.45) . In cont = 0.45) . In adul = 0.45) . However = 0.45) . On the  = 0.45) . O’Neill = 0.45) . AFP can = 0.45) . Convers = 0.45) . Neverth = 0.45) . In term = 0.45) ,67,68.IC-GCTs account for 0.3–3.4% of childhood CNS tumors in North America and Europe, but the incidence rises to 15% in East Asia . IC-GCTsThe occurrence of SCT has a frequency of 1 in every 35,000–40,000 births. The most common type is mature teratoma; however, 11–35% of cases involve a mixture of a malignant component, most commonly YST or EC . Unlike YST is the most common malignant germ cell tumor in children and is histologically composed of yolk sac mesenchymal cells. Approximately 70–90% of YST cases secrete AFP ,84, but Histopathologically, germinoma is the most primitive form of germ cell tumor, capable of differentiating into YST or immature teratoma. It consists of cells that produce AFP (from YST) and b-HCG (from choriocarcinoma) . GerminoA rare, unilateral mixed-sex cord-stromal tumor. It is mainly found in young women (75% of cases), but the youngest known case was 9 months old . ApproxiCareful consideration should be given to using AFP levels as a basis for clinical decisions in neonatology since they are physiologically elevated from birth until the first year of life.In young children, elevated AFP levels can mask the presence of certain genetic diseases, liver regeneration in chronic diseases, and tumorigenesis processes.In pediatric cases, AFP remains a valuable marker for liver tumors and GCTs that involve tissue elements derived from the yolk follicle. Monitoring AFP levels in children with various chronic liver diseases can help predict the early onset of HCC.It is necessary to develop new pediatric reference ranges for modern AFP diagnostic tests based on current laboratory techniques.


The purpose of this study was to investigate the mechanism of sanguinarine (SAN) against nasopharyngeal carcinoma (NPC) by means of network pharmacology, molecular docking technique, and experimental verification.The SAN action targets were predicted using the Swiss Target Prediction database, the related NPC targets were determined using the GEO database, and the intersection of drug and disease pathway targets were considered to be the potential targets of SAN against NPC. The target-protein interaction network map was constructed using the STRING database, and the core target genes of SAN against NPC were obtained via topological network analysis. “R” language gene ontology (GO) function and Kyoto encyclopedia of genes and genome (KEGG) pathway enrichment analyses were used to dock the core target genes with SAN with the help of AutodockVina. Cell proliferation was detected using MTT and xCELLigence real-time cell analysis. Apoptosis was identified via Hoechst 33342 staining, JC-1 mitochondrial membrane staining, and annexin V-FITC/PI double fluorescence staining, while protein expression was quantified using western blotting.A total of 95 SAN against NPC targets were obtained using target intersection, and 8 core targets were obtained by topological analysis and included EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1. Gene ontology enrichment analysis identified 530 items, and 42 items were obtained by Kyoto encyclopedia of genes and genome pathway enrichment analysis and were mainly related to the PI3K/AKT, MAPK, and p53 signaling pathways. Molecular docking results showed that SAN had good binding activity to the core target. SAN inhibited the proliferation of NPC cells, induced apoptosis, reduced the expression levels of survivin and Bcl2, and increased the expression levels of Bax and cleaved caspase-8. It also decreased the expression levels of the key proteins p-c-Raf, p-MEK, and p-ERK1/2 in the MAPK/ERK signaling pathway in NPC cells.SAN inhibits the proliferation and induces the apoptosis of NPC cells through the MAPK/ERK signaling pathway. Scholars believe that the occurrence of NPC is the result of a combination of factors, which is related to family genetic susceptibility, EB virus infection, environmental factors, and eating habits. The most common site of NPC is in the pharyngeal recess. Because of its hidden location and the lack of specificity of early symptoms, NPC is not diagnosed in time, its metastasis rate is high, and most of NPC cases are poorly differentiated carcinomas, making its prevention and treatment more difficult. NPC is sensitive to radiation and radiotherapy is the first choice for its treatment. Simultaneous chemotherapy can enhance the antitumor effect and reduce the metastasis and recurrence rates.,3 However, radiotherapy and chemotherapy have limitations. The side effects caused by this treatment, such as dry mouth, mucosal injury, and nerve injury, result in significant levels of pain and lead to a decline in quality of life. Local recurrence and metastasis are still the main causes of failure in the treatment of NPC. More and more studies have shown that natural small molecular compounds demonstrate efficacy in the treatment of tumors with low levels of side effects and high safety. The discovery and application of these drugs are expected to provide a new treatment plan and perspective for tumor treatment. Sanguinarine (SAN) is a phenanthrene alkaloid extracted from plants, such as Macleaya corydalis and Corydalis. Studies have found that SAN has a wide range of pharmacological effects, which can activate the AMPK/Smad3 signaling pathway to promote MC3T1-E1 cell differentiation, stimulate bone growth, treat osteoporosis, inhibit activation of the P38 MAPK signaling pathway, relieve neuropathic pain, reduce the pathogenicity of Magnaporthe oryzae, inhibit the MAPK signaling pathway, and affect the release of inflammatory mediators.At present, more and more attention has been paid to the antitumor research on SAN, which covers many aspects, such as signaling pathways and microRNAs.,11 SAN has therapeutic effects on lung adenocarcinoma, breast cancer,,14 ovarian cancer, thyroid cancer, colorectal cancer, and gastric cancer. It also inhibits the proliferation of non-small cell lung cancer, blocks the cell cycle, and induces apoptosis by changing the content of STAT3. In addition, its mechanism is related to ferroptosis. Sarkhosh-Inanlou et al have reported that SAN reverses the sensitivity of cisplatin-resistant ovarian cancer cells to cisplatin by reducing the glutathione content. S. Zhang et al have shown that SAN has an anti-ovarian cancer effect via the long noncoding RNA CASC2/EIF4A3 axis, NF-κB, and PI3K/AKT/mTOR signaling pathways. It also affects the viability of triple negative breast cancer cells through CCL2 and the IKBKE/NF-κB/ERK1/2 signaling pathway. In hypoxic conditions, SAN inhibits the expression of HIF-1α in hepatoma cells, further inhibiting the secretion of TGF-β and epithelial stromal transformation induced by hypoxia. It has been reported that SAN can inhibit the proliferation, apoptosis, and invasion of NPC cells through the mTOR signaling pathway. However, research on its mechanism is still not comprehensive enough, which hinders its in-depth study and clinical application.Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the head and neck. Its incidence has obvious regional characteristics, mostly in southeast Asia, and a high incidence in southern China, affecting more males than females.,24 Based on this, the purpose of the present study was to use network pharmacology and molecular docking technology to explore the anti-NPC mechanism of SAN and to verify its antitumor activity and mechanism against NPC through experiments.Network pharmacology is a new strategy that integrates systems biology, high-throughput screening, and computer technology to study the complex relationship between “drug-target-disease” and accelerate the identification of drug targets. Molecular docking technique is a method to evaluate the interaction between organic small molecular ligands and target proteins, which can be used to determine the binding sites and binding forces of small molecules in natural products and target proteins.http://bionet.ncpsb.org.cn/ batman-tcm/index.php/Home/ Index/ index) as ID: 5154 and the target with score of > 20 was selected. The molecular structure was uploaded to the PharmMapper database (http://lilab-ecust.cn/pharmmapper/). Results with Norm Fit of >0.5 and z-score of >0 were selected, and the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) was searched using “Sanguinarine” in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (https://tcmsp-e.com/tcmsp. php) database and the corresponding formula for its molecular structure. The above results were combined to obtain the SAN targets. The results were corrected using the reviewed target from Swiss-Prot and Human in Uniprot data.SAN was uploaded to the pubchem BATMAN database  to obtain the intersection target of SAN in the treatment of NPC.Transcriptome datasets related to NPC were selected from the GEO database and included GSE68799, GSE118719, and GSE134884 (Table Interactions between target–target function-related proteins were obtained from the mapped intersection targets for NPC SAN treatment in the STRING (version 11.5) database. The selected minimum required interaction score was >0.700 and the PPI network map of target interaction and its tsv.data were obtained. The topological parameters, such as the median degree of freedom and the maximum degree of freedom in the network, were analyzed using NetworkAnalyzer in Cytoscape_v3.7.1. The core targets were selected based on 2 times the degree.P-value cutoff for enrichment was .05.R packages including “ClusterProfiler” (version 4.1.3) were used to analyze and visualize the GO and KEGG pathway enrichment of the module and core targets. Gene annotation information came from “org.Hs.e.g..Db” and https://pubchem.ncbi.nlm.nih.gov/), and the corresponding protein structure was extracted from the PDB database (https://www.rcsb.org/). The SAN structure was optimized using minimum energy MM2 position 3D Draw module in Chem Bio Office software and transformed into PDBQT using Raccoon software. Autodock Vina software-supporting tool MGLTools 1.5.6 was used to evaluate the proteins, hydrogenation, and Gasteiger charge, as well as merge nonpolar hydrogen and to perform other operations. The results were saved in pdbqt format to prepare for ligand docking.Molecular docking can predict the binding ability of proteins to small molecules. The SAN structure was obtained from PubChem database  twice. Then, 1 mL of Hoechst 33342 staining solution was added and incubated for 20 minutes in the dark. Next, the staining solution was removed, and the excess dye was washed with PBS. Finally, the cells were imaged using a multi-functional cell imaging system.The cells were digested, resuspended at a concentration of 1 × 105 cells/mL, and 1 mL/well of the cell suspension was added to a six-well plate. After the cells were attached, 2 mL of the drug-containing culture medium was added. The culture medium was removed after 24 hours and washed twice with PBS. Then, 1 mL of the JC1 staining solution was added and incubated for 20 minutes in the dark. The staining solution was then removed and the excess dye was washed out with PBS. The cells were imaged using a multi-functional cell imaging system.The cells were digested, resuspended at a concentration of 1 × 10After the intervention with SAN, the supernatant was collected in an Eppendorf (EP) tube and the adherent cells were digested with trypsin without EDTA. Then, 2 mL of the whole culture was added to terminate the digestion, the cell suspension was collected in an EP tube containing the supernatant, and the supernatant was discarded and transferred to a new 1.5-mL EP tube with PBS solution. The supernatant was removed by centrifugation and 100 μL of binding buffer was gently added, followed by 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) staining solution while avoiding light exposure for 15 minutes. Then, 100 μL of binding buffer was added to stop the staining reaction. The apoptosis rate was detected using a double fluorescence cell analyzer.After SAN treatment for 24 hours, the total protein was extracted and quantified. The protein samples were then added to the gel electrophoresis system, concentrated at a voltage of 40 V in the gel, and pressurized to 80 V after entering the separation gel at 200 mA of constant current. 2.5 hours transfer film and 5% skimmed milk powder closed for 1 hour. The primary antibody was incubated overnight at 4 ºC, and the secondary antibody was incubated at room temperature for 1.5 hours. The film was scanned using a two-color infrared fluorescence imaging system, and the band signal value was analyzed using ImageStudio.P < .05 was considered statistically significant. Results were graphically presented using GraphPad prism 8.0 software.SPSS 26.0 statistical software was used to process all of the data in the present study. The experimental data followed a normal distribution and were expressed as the mean ± standard deviation. Univariate analysis of variance was used for univariate design and comparisons of measurement data among multiple groups. The LSD method was used for multiple comparisons. A total of 7852 disease genes were extracted from the GEO database. Drug targets were searched in the SwissTargetPrediction database. A total of 449 SAN targets were obtained by removing duplicates and corrected using the Uniprot database. As a result, 95 intersection targets were identified Fig. A. At theThe mapped intersection protein was introduced into the Cytoscape 3.7.1, and the topological parameters of SAN anti-NPC target and function-related protein interaction network were calculated. The median degree of freedom for the target was 4.588, and the maximum degree of freedom was 19. Therefore, the range of core target screening conditions was set to 10–19, and 8 core targets were obtained, including EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1  of SAN and Cis (4 μg·mL−1) for 12, 24, 36, and 48 hours, the proliferation of CNE2 cells was significantly inhibited , MMP9, and SERPINE1 was positively correlated with NPC metastasis and promoted the proliferation and invasion of NPC cells.–34 CDK1 plays an important role in the development of NPC. The results of molecular docking showed that there is good binding between SAN and the core targets, suggesting that the anti-NPC effect of SAN is related to the regulation of these core targets. The present study experiments showed that SAN can inhibit the proliferation and induce apoptosis of NPC cells. Bax and Bcl2 are the key factors determining apoptosis in tumor cells. The present results showed that SAN can reduce the expression of antiapoptotic protein Bcl2 and increase the expression level of proapoptotic protein Bax. Caspase-8 mainly mediates the mitochondrial apoptosis pathway. Prabhu et al and others have shown that SAN can promote the expression of cleaved caspase-8 and stimulate mitochondrial apoptosis in non-small cell lung cancer. Similarly, the present study experiments showed that SAN can increase the expression of cleaved caspase-8 in NPC cells, suggesting that SAN-induced apoptosis in NPC cells may be related to the mitochondrial pathway.In this study, 95 SAN targets against NPC were obtained based on the reverse targeting strategy. Eight core targets were identified via topological analysis, including EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1. Recent studies have shown that EGFR plays an important role in promoting cell proliferation, cell protection, and apoptosis-mediated differentiation, and that EGFR expression is closely related to NPC prognosis. indicating that SAN can inhibit proliferation and induce apoptosis of NPC cells via the MAPK/ERK signaling pathway.GO enrichment analysis resulted in 530 items, and KEGG pathway enrichment analysis showed 42 items, which were mainly related to the PI3K/AKT, MAPK, and p53 signaling pathways. The MAPK signaling pathway is involved in the occurrence and development of NPC. The effect of SAN on the MAPK/ERK signaling pathway was verified and revealed that SAN can inhibit the expression level of the key proteins in the signaling pathway, including p-c-Raf, p-MEK, and ERK1/2, which was consistent with previous studies,The above analysis and argument indicate that sanguine is suitable for the preparation of anti-nasopharyngeal carcinoma drugs. Although the inhibitory effects of sanguine on nasopharyngeal carcinoma proliferation and induction of apoptosis have been clarified, the process of inducing apoptosis is still unclear. And this study has only completed network pharmacology and cell experiments, therefore, in further research, it is necessary to improve in vivo experiments and the entire regulatory network.SAN can inhibit the proliferation and induce the apoptosis of NPC cells via the MAPK/ERK signaling pathway. The core targets of this mechanism action include EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1.Data curation: Wen-Qing Zhang, Ting Lin.Formal analysis: Fang-Liang Zhou.Funding acquisition: Jing-Ying Fan.Methodology: Ying-Chun He, Hong-Jian Shi.Project administration: Ying-Chun He.Resources: Xi-Ran Hu.Writing – original draft: Jing-Ying Fan, Jie Liu.Writing – review & editing: Le Tang, Hong-Jian Shi.


Camelus dromedarius during the breeding season.The rutting period imposes a stressful condition on male camels, which results in elevated serum cortisol levels and alterations in their sexual behavior. Therefore, the current work was carried out to investigate the effect of mineral-vitamin premix supplementation on behavior, reproductive performance, hormones, serum oxidative stress profile, and other serum biochemical parameters of Camelus dromedarius were divided into two groups, a control group (n = 7) and a mineral-vitamin premix group (n = 7). The present study lasted for 95 days during the rutting period (1st February to 5th May). Each camel in the premix group received a daily diet of 50 g of mineral-vitamin premix throughout the whole rutting period, during which the frequencies and durations of the following behaviors: maintenance, posture, aggressiveness, and sexual activity were collected every 20 min. At the end of the study, blood samples were collected.Fourteen mature, fertile male P < 0.05) maintenance (feeding and rumination), standing, and overall sexual desire-related behavior frequency, besides more times (P < 0.001) for rumination, standing, walking, and lying while showing lower (P < 0.001) frequencies of overall aggressive behaviors than the control group. The serum concentration of malondialdehyde, nitric oxide, cortisol, blood glucose, and urea evidenced a significant decrease in the premix group compared with the control one, while significantly elevated levels of reduced glutathione, testosterone, total antioxidant capacity, triiodothyronine, and thyroxin, total protein, albumin, globulin, calcium, phosphorus, potassium, and magnesium were recorded in the premix group in comparison with the control.Results revealed that the premix group showed higher (It could be concluded that daily dietary supplementation of 50 g of mineral-vitamin premix to male camels during the breeding season is necessary to overcome the oxidative stress and serum cortisol concentration with a subsequent decrease in aggressive behavior and improvement to testosterone level in blood, body condition score and body weight gain. Camelus dromedarius population is raised in arid and semi-arid regions, including Egypt, due to the unique physiological characteristics that enable them to survive and perform in such harsh environments , performance , serum oxidative stress profile , hormone levels , and other serum biochemical parameters .Finally, as a result of the steady increase in human population numbers and the insufficient food production in Africa and parts of Asia, it is compulsory to develop suitable systems for livestock production, accompanied by the optimal and effective utilization of available and untapped native resources . In thisThe protocol of this experiment was approved by Zagazig University's institutional animal care and use committee (ZU-IACUC) under the number (ZU-IACUC\2\F\80\2023).Camelus dromedarius aged 5–8 years, that reached puberty at 3–4 years  in Egypt. This study lasted for 95 days during the rutting season (1 February-5 May). Semi-covered areas with sand floors (15 m2/camel) were used as housing  with a constant quantity and quality of feed during the experiment period with free access to fresh water. The quantity was approximately 3% of their live weight in dry matter, while the quality was 40% concentrate (11% protein) and 60% hay. The recommended feeding schedule was 8–9 a.m. concentrate, 11 a.m.−12 p.m. hay, and 4–5 p.m. concentrate.n = 7 per group): the control group was fed the basal diet only, and the premix group received the control diet plus 50 g of mineral-vitamin premix per camel daily throughout the whole rutting period. The schematic cartoon of this study is illustrated in Fourteen male dromedary camels were used  were recorded (1 h twice a week (Saturday and Tuesday) as 20 min in the morning (8:20–8:40), midafternoon (12:20–12:40), and afternoon (3:20–3:40) for 10 weeks, which were analyzed using the focal sample technique [3 times per day\2 days (Saturday and Tuesday)\week = 1 h × 2 days × 10 weeks × 7 camels = 140 h per each group] . The behVisual observation was done daily after 2 h of feeding for the scoring of trough leftovers. The scores were assessed on a scale from 1 to 5, according to Stadler et al. .The average of initial and final body weights was weighted by kilograms (kg), and the average weight gain was calculated by the difference between them.via visual assessment from an interval space of 2–3 m far from the camel, while hump size was assessed by an approximate determination of the dorsal line proportion occupied by the body structure as illustrated by Robinson  for the separation of the clear serum, which was carefully collected in Eppendorf tubes and stored at −80°C until hormonal and biochemical analysis.At the end of the rutting period, the blood specimen was taken from the jugular vein and placed into plain vacuum tubes. Then, the tubes were left for 20 min in an inclined position at room temperature. After that, the tubes were kept in the fridge to evade glycolysis and until there was complete blood clot retraction. Following that, the samples were put in the centrifuge for 10 min at 3,000 rpm . Oxidative stress variables were assayed using a semi-automatic photometer  according to the manufacturer's instructions.Oxidative stress variables, including serum malondialdehyde (MDA), total antioxidant capacity (TAC), reduced glutathione (GSH), and nitric oxide (NO), were analyzed Serum concentrations of cortisol, triiodothyronine (T3), thyroxin (T4), and testosterone were investigated using commercial ELISA kits . The ELISA reader was used  at 450 nm.via the colorimetric method using the commercial kits. Globulin levels and the albumin/globulin (A/G) ratio were calculated , urea , creatinine, albumin, calcium phosphorus, magnesium, and potassium , were investigated lculated . All part-tests using the SPSS software program version 23.0  frequencies of maintenance  in the premix group in comparison with the control group. Mineral-vitamin premix supplementation significantly affected the sexual and aggressive behaviors of male dromedaries, where the premix group expressed a higher (P < 0.05) frequency of some sexual desire-related behaviors, including extruding gulla and poll gland secretion. On the other hand, there was a significant decrease in the frequency of aggressive behaviors compared with the control group, as shown in The effects of mineral-vitamin premix supplementation on maintenance behavior (frequency and duration), posture, and aggressive and sexual behaviors of male The scoring of trough leftovers, average final body weight, body gain, and body condition score were significantly elevated in the premix-treated group in comparison with the control one .Both serum MDA and NO evidenced a significant decrease in the premix-treated group as compared with the control group. On the other hand, both serum GSH and TAC were significantly increased  in the p3, and T4 levels. However, it had a significantly lower serum cortisol level than the control  in serum TP, albumin, Ca+2, P+3, magnesium, Mg+2, and K+ in the premix group compared with the control (P < 0.05) was recorded in blood glucose and urea in the premix group compared with the control group , and welfare of the animals. Scoring is based on determining the amount of fat and muscles over and around the vertebrae . In addiThe obtained data revealed a significant increase in feeding, rumination behaviors (frequency and duration), final body weight, body weight gain, and body condition score, while there was a significant decrease in the leftover score in the premix group as compared with the control. This result matches that of Faye et al.  who recoMoreover, feed efficiency and average daily gain had an irreversible relation to protein and energy levels in the diet . FurtherIn addition, data in the current study showed significantly increased activity in standing and walking in the premix group as compared with the control group, which may be a result of displaced animals waiting for access to the feed  and cameAggressive behavior is considered a functional form of social behavior that can be seen in nearly all animals, and it has many functions, such as protecting the home range and its resources against intruders and/or maintaining the flock's social hierarchy .Camelus dromedarius) and recorded that cortisol levels significantly increased (17.21 ± 2.96 μg/dl = 172.1 ± 29.6 ng/ml) in camels that were transported by truck  between mountains as compared with control (3.35 ± 0.38 μg/dl = 33.5 ± 3.8 ng/ml).Rut season in male camels constitutes a stress period that is accompanied by a general state of increased irritability and aggressiveness, which is associated with an elevated serum cortisol level , 47. DueIn this regard, the results of the current study showed a significant decrease in overall aggressive behavior frequency in the premix group in comparison with the control group, which was confirmed in the laboratory findings of the current study by decreased MDA, NO, and cortisol serum levels and increased serum concentrations of GSH, TAC, and testosterone in the premix group as compared with the control group.Thus, oxidative stress results when reactive substances, including reactive nitrogen, oxygen, and chlorine species, exceed the available antioxidant capability in the body. If the antioxidant systems in the animal body become incapable of adequately coping with enhanced reactive oxygen species (ROS) amounts and with the subsequent series of reactions of harmful macromolecule modifications  , 55, as Regarding the obtained data, there were significant reductions in NO and MDA. On the other hand, GSH and TAC concentrations were significantly elevated in the premix group in comparison to the control group; this could be due to the antioxidant effects of vitamins E, C, and A contained in the mineral-vitamin premix –61. Thus2O2, and phospholipid membrane oxidation which are essential for protection from oxidative stress  between the premix group and the control group. Furthermore, it could be due to vitamin A, which is one of the components of the premix and is very important in many physiological functions, particularly reproduction, growth, differentiation of tissues, and maintenance of the epithelium. It has a natural antioxidant property and is necessary to maintain the integrity of the tissue lining the GIT and genitourinary tract, as well as to contribute to ensuring the optimal functioning of the immune system , 78.The rutting period compels a stressful condition in male camels, which is associated with elevated serum cortisol levels accompanied by decreased testosterone hormone concentration in the blood. Therefore, the feeding of minerals and vitamins to male camels during rut season is necessary to overcome the oxidative stress to which the animal is subjected, with a subsequent decrease in cortisol level followed by a decrease in aggressive behavior and an increase in testosterone hormone in the blood, in addition to a significant increase in feeding, rumination, and sexual behavior and an improvement in body condition score.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was approved by the experimental protocol regarding the care and management of the camels was approved by the ZU-IACUC Committee under the number (ZU-IACUC\2\F\80\2023). The study was conducted in accordance with the local legislation and institutional requirements.NN, RE-S, and GA-R conceived, designed, performed the experiments, and analyzed the data. WS contributed to the analysis tools. NN, RE-S, HG, and WS wrote the manuscript. IR, FZ, and ZF revised and edited the manuscript. All authors have read and agreed to the published version of the manuscript.


Among our panel, we identified several promising inhibitors with IC50 values below 200 μM, with our strongest inhibitor – LPRDSar – having an IC50 of 18.9 μM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 μg mL−1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed  S. aureus.A series of peptidomimetic Sortase A inhibitors is reported. These compounds show inhibition activity of the Sortase A enzyme and efficiently prevent biofilm formation of  The mechanisms of these conventional antibiotics are mainly associated with the prevention of bacterial growth (bacteriostatic) and inducing bacterial cell death , which is typically achieved by inhibiting vital bacterial metabolism such as nucleic acid, protein and cell wall synthesis.6 Different types of virulence factors have been identified, such as surface proteins, toxins, hydrolytic enzymes and capsules.6,7 Targeting these virulence factors, instead of conventional targets of antibiotics, has gained increasing interest as these treatments have no bactericidal activity and no effect on bacterial cell growth, but interfere mainly with bacterial mechanisms that initiate the infection.4 In fact, antivirulence treatments disarm pathogenic bacteria rather than killing them and, as a result, employ low selective pressure to induce the development of antibiotic resistance.3,8,9Bacterial virulence factors are molecules that enable bacteria to colonize and infect the host, as well as increase their capacity to damage the host tissues.10 playing a critical role in the formation of biofilms which protect the pathogen from the host immune system.11 Inhibition of the bacterial adhesion process has been recognized as a promising anti-virulence approach.12 Pili in Gram-positive bacteria are structural protein motifs that are covalently linked to the peptidoglycan layer.13 These cell wall anchored (CWA) proteins play a major role in adhesion since they are used to target the host's extracellular matrix proteins such as collagen, fibrinogen and fibronectin. A subfamily of CWAs, the MSCRAMMs , are covalently linked to their cell wall peptidoglycans by unique cysteine transpeptidases called sortases.13 Sortase enzymes are ubiquitous in Gram-positive bacteria and have an important role in bacterial virulence. Among these, Sortase A (SrtA) is considered a housekeeping sortase and is of great interest as a potential target for antivirulence treatments.14Bacterial adhesion to the host tissues is the first crucial step for infection and colonization,14 SrtA recognizes MSCRAMMs by their sorting signal consisting of an LPXTG motif, where X represents any amino acid.14 The anchoring process is initiated by recognition of the LPXTG motif, followed by a transthioesterification reaction, which cleaves the peptide bond between the threonine and the glycine residue of the sorting motif, resulting in the formation of a thioester acyl-enzyme intermediate  infections pose a huge threat to modern-day clinics, with high reported morbidity and mortality rates.19 Therefore, novel treatments for these infections are in high demand, and inhibition of SrtA could serve as a long-term solution, due to its antivirulence activity. Septic arthritis, a severe infection of synovial fluid and joint tissues with nearly 50 000 annual cases in Western Europe and the US caused by different bacteria, viruses or fungi, has been directly linked with the activity of S. aureus SrtA.20–23 It was shown that SrtA-knockout mice infected with S. aureus strains did not show severe symptoms of septic arthritis, with increased survival rates and significantly lower weight loss reported. Interestingly, knockout of the closely related Sortase B did not show such an effect, pinpointing SrtA as an essential factor leading to the advent of this disease.24 Septic arthritis is particularly prevalent in children and treatments commonly rely on conventional antibiotics, with an often complicated recovery.25 Therefore, specific and effective treatments are highly desired, of which SrtA inhibition is a prime candidate.Several diseases have been directly linked with the activity of SrtA so that inhibition of this enzyme provides potential treatment options against these diseases, making it a prime target for drug development. Over 25 years ago it was already identified that typical hospital bacteria such as 5 Among the peptide-based inhibitors, only modest inhibition of SrtA efficacy was reported to date.4 Most compounds are based on the LPXTG sorting signal and replaced the Thr–Gly scissile bond with a variety of functional groups. Reversible inhibitors containing a phosphonic ester bond were shown to have poor inhibition.26 Several potential covalent inhibitors were also developed, containing chloromethyl ketone, diazo ketone, vinyl sulfones, sulfhydryl, cyanoalkene groups or a thiol-containing threonine analog, however, none of these peptides displayed exceptional inhibitory potential.27–30 Finally, a virtual screening-based approach was employed to identify the strongest peptidomimetic SrtA inhibitor to date.31 The peptide PEG2000-LPRDA-NH2 was reported to have an IC50 of 10.61 μM and showed inhibition of biofilm formation by interaction with fibronectin and by invasion into the host tissues in S. aureus treated with the oligopeptide.31To date, several SrtA inhibitors have been identified, derived from different types of natural products, small organic molecules and peptides.Still, none of the previously published compounds are currently used clinically and among the peptidomimetic inhibitors, only high micromolar inhibition has been achieved. Furthermore, most of the tested molecules are covalent inhibitors, which are prone to have multiple off-target effects with other cysteine proteases in the body. Therefore, we set out to systematically develop potentially potent peptide-based antivirulence compounds as SrtA inhibitors.2000-LPRDA-NH2 structure, reported as a SrtA inhibitor by Wang et al. with an IC50 of 10.61 μM  group, which is composed of two PEG units, was added to either the C- or N-terminus. Compound 4, containing the D4C substitution, has the potential to act as a covalent inhibitor by forming a disulfide bond with the active site Cys134 of the SrtA enzyme. In 5 Ala5 was substituted by N-methylglycine , where the substrate cleavage position at the T–G amide bond is blocked by methylation  was inspired by the PEG10.61 μM .31 In orhylation .32 The substrate contains a T–G scissile bond which is cleaved by SrtA to form the virulence factor. To increase the stability of this bond against the proteolytic cleavage of the enzyme, the T–G amide bond was modified by N-methylation in compound 6 and a G5P mutation in compound 7. Peptide 8 was designed to mimic part of the natural substrate of the enzyme, KSELPETGGEE. Finally, to test the affinity of the enzyme to the substrates' reversed sequence, peptide 9 was introduced.The structure of the second series of peptidomimetic inhibitors (6–9) was based directly on the LPETG substrate sequence .32 The s27–30 where often a Cbz group was introduced at the N-terminus and our own computational studies (details are discussed later in the manuscript), we sought to more systematically investigate the influence of the introduction of steric bulk at the N-terminus. To this end, a third series of peptidomimetic inhibitors 10–14 was developed, carrying varying degrees of bulk, with an acetyl group in 10 being the smallest and a 2-naphthoyl in 13 the largest. Furthermore, the importance of aromaticity was investigated by including the benzoylated peptide 11 and a peptide bearing a cyclohexyl group in peptide 12.Based on previous peptidomimetic inhibitors of SrtA,cis- or trans-conformation, where most often the trans-conformation is energetically favored, however, the cis-conformation can be of importance for biological activity.33,34 As earlier shown by us and others, 1,2,3-triazoles can be used as amide-bond surrogates, where the 1,4-disubstituted triazole mimics the trans-conformation and the 1,5-disubstituted triazole the cis-conformation, furthermore.33,35 Additionally, the triazoles are also more stable to proteolytic degradation.36 Both the 1,4-disubstituted triazole and 1,5-disubstituted triazole are synthetically easily accessible by CuAAC and RuAAC, respectively.37,38Finally, the fourth series of inhibitors, peptides 16 and 17 were prepared to investigate the influence of the orientation of the peptide bond between the N-terminal leucine and proline residue in the inhibitor peptides. Peptide bonds can exist in either the All the peptides 1–17,  were syn39 In the present work, the Abz-LPETGK(Dnp)-NH2 substrate was used to monitor the transpeptidation activity of SrtA and its kinetic parameters were determined.40 SrtA utilizes the substrate Abz-LPETGK(Dnp)-NH2 and H-Gly3-OH, producing Abz-LPETGGG-OH and GK(Dnp)-NH2 as depicted in Fig. S32a.†In the bacterial cell membrane, SrtA cleaves the Thr–Gly scissile bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 moiety of lipid II .39 In th2 peptide and the consumed substrate peak over time. High-throughput applications of this method for analysis of SrtA inhibition, substrate specificity, and kinetic mechanism are also feasible.41 We used a modified protocol from Kruger et al. to determine the kinetic parameters of recombinantly expressed SrtA  with a varying concentration of its Abz-LPETGK(Dnp)-NH2 substrate, Michaelis–Menten kinetics could be applied to obtain the kinetic parameters for the SrtA enzyme  through analyzing the peak of the released GK(Dnp)-NHFig. S32c. From th2 substrate was used as a positive control and showed that around 26% of the substrate was cleaved by the enzyme after 4 hours . The Abz-LPETGK(Dnp)-NH Fig. S33. This ac Fig. S33.2  were tested for SrtA inhibition at 200 μM in HEPES buffer , making it the strongest inhibitor identified in the first series, while surprisingly, the introduction of Sar in 6 did not result in effective inhibition amongst the second series. 7, on the other hand, was found to be the strongest inhibitor among all four tested series (76%), indicating that replacing the glycine of the scissile T–G bond by either Sar or Pro can be an effective strategy to achieve SrtA inhibition. 8 possessing a high similarity to the natural substrate was not able to efficiently compete with the Abz-LPETGK(Dnp)-NH2 substrate with an extent of inhibition of 18%. 9, bearing the reverse sequence of the sorting motif, had moderate inhibitory potency (38%), indicating a loss of recognition when the sequence is reversed.In order to screen the activity of SrtA inhibitors, a FRET-based functional assay was used which employs an internally quenched fluorescent (IQF) substrate, Abz-LPETGK(Dnp)-NHFig. S37a.5 The syS buffer . 5-, furthermore, precursor 15, lacking the locked amide bond, showed a similar potency (24%).Among the N-terminally modified peptides, acetylation did not have a noticeable effect on inhibitory potential, while surprisingly the Cbz-modified peptide was the worst inhibitor in this series. Benzylation of the LPRDA sequence increased the inhibition rate in 11 to 37%, while the non-aromatic counterpart 12 was less active. Due to the incorporated 2-naphthoyl group in 13, which was an inherent fluorescent signal, oversaturation of the detector was reached at 200 μM. Therefore, the single point inhibition was carried out at 10 μM, resulting in a substantial inhibition at 47%. Surprisingly, 14, carrying an N-terminal Cbz-group, which was previously used for covalent peptidomimetic inhibitors of SrtA,Taken together, these results suggest that the tested peptidomimetic compounds can act as competitive inhibitors at different rates ranging from 17% up to 76% at 200 μM inhibitor concentration. Among the initial panel, two peptides (5 and 7) showed strong inhibition, both of which contain a non-scissile bond in the C-terminal amino acid, in the form of sarcosine in 5 and proline in 7. Furthermore, benzoylation of the N-terminus proved to slightly increase the inhibitory potential as well.Based on our initial series of peptidomimetic SrtA inhibitors and the understanding gained from our computational analysis (discussed later in the manuscript) a new generation of peptides was designed to more efficiently target SrtA. Firstly, 5 and 7, which showed good initial inhibition, were benzylated to potentially gain a synergistic effect with the increased inhibition observed for the benzylated peptide 11, to obtain peptides 18 (BzLPRDSar) and 19 (BzLPETP). It was observed that the incorporation of sarcosine in the LPRD series resulted in efficient inhibition, while the incorporation of proline in the LPET sequence had the same effect. To complete the range of combinations, peptide 20 (LPRDP) and the benzylated variant 21 (BzLPRDP) were added to the panel as well . FurtherFirstly, all second-generation peptides were analyzed regarding their stability towards SrtA, which showed no detectable degradation in any of the samples Fig. S36. Hencefo50 values were accurately determined for the most potent inhibitors from the initial screen, namely 5, 7, 18, 22 and 23, as well as 24 to directly compare the influence of benzoylation, by varying their concentration between 1 μM and 1500 μM while measuring SrtA inhibition  displayed an IC50 below 100 μM, with the strongest inhibitor at 18.9 μM for LPRDSar, indicating that this sequence is the most promising for in vitro inhibition of the S. aureus SrtA enzyme activity. Interestingly, when comparing 23 and 24, benzoylation of the sequence resulted in a slight improvement of inhibitory potential for the benzoylated peptide, while for 5 and 18 the effect was not present. In summary, our series of peptidomimetic SrtA inhibitors appear to be the most potent compounds in this class so far and therefore all six tested compounds were further evaluated for their activity against pathogenic S. aureus in growth inhibition studies. Due to the previously mentioned detector saturation, 13 was not evaluated for an IC50 measurement. Nevertheless, its significant inhibition at 10 μM suggests an IC50 in the range of our strongest inhibitors. Therefore, 13 was evaluated for activity against S. aureus.Subsequently, IChibition . All pepS. aureus bacteria to assess the peptidomimetic inhibitors for their ability to inhibit bacterial growth. Firstly, our compounds were incubated with S. aureus bacteria in a 96-well plate in Tryptic Soy Broth (TSB) medium, while we also included the reference compound used in the single-point in vitro inhibition studies. So far, the covalent binding thiadiazol reference compound was not tested in this respect, but was one of the most promising compounds identified in an earlier screening campaign.42 The inhibitors were added at various concentrations, ranging from 2 μg mL−1 to 128 μg mL−1, and the absorbance at 600 nm was measured as an indication of their optical density (OD600) and by that the bacteria's ability to propagate in liquid media. In the negative control experiments, the bacteria consistently reached an OD600 of 0.6 between 6 and 12 hours  medium in a 96-well plate coated with rabbit plasma, to allow the bacteria to form biofilms. After 12 hours of incubation with the peptides, the samples were stained with crystal violet (CV) and then solubilised in ethanol to quantify the biofilms by measuring the absorbance at 595 nm  substrate binding site for our anticipated docking approach . In brief, 2KID was aligned with 7S51 and the binding peptide Abz-LPATAG from 7S51 was transferred to 2KID keeping its original binding orientation. Then the ligand was transformed into LPRDSar (ligand 5 in this study) followed by a short refinement molecular dynamic simulation. Thereby, the ligand was fixed in the binding pocket through distance restraints, which mimic the hydrogen bonds between the ligand and the protein in 7S51, allowing the protein to adapt. With respect to our anticipated docking study the highly flexible binding pocket of SrtA is especially problematic, which has been shown by others and also agrees with our own experience from other computational studies.42,46,47 The conformational flexibility of the substrate binding pocket was already observable in our short refinement simulation  was use Fig. S41, showing Fig. S41.S. aureus structures and in depth analyzed  but also showed large deviations of up to 2.75 kcal mol−1  when they are bound in an inverse orientation  and a previously reported sequence (LPRDA).31With the emergence of resistant bacteria, targeting bacterial virulence factors, instead of developing compounds with bactericidal effects, has attracted a lot of attention. If antivirulence treatment is effectively applied, the pathogen becomes unable to form a threat, without killing them, thus leading to a low selective pressure to develop resistance against the antivirulent treatment. Amongst the virulence factors, SrtA is a particularly promising target. SrtA is a membrane-bound transpeptidase enzyme, important for bacterial adhesion to host cells. Previously, inhibition of SrtA in 50 value of below 200 μM, with the compound LPRDSar having the lowest IC50 of 18.9 μM. Compounds based on the LPRDA motif seemed to work most effectively, with the introduction of either a non-scissile moiety at the C-terminus, or inclusion of some form of aromaticity in the sequence generally enhancing the inhibitory potential. With these results, our compounds are the strongest in vitro peptidomimetic compounds known to date. Furthermore, the six most effective compounds were tested for their potential to inhibit the growth of S. aureus bacteria. Here, it was found that compounds containing a phenyl moiety exclusively showed an antivirulent effect, present in the form of a phenylalanine residue or a benzoylated N-terminus. Interestingly, compounds lacking this showed no activity towards the bacteria at all, exemplified by LPRDSar being the strongest in vitro inhibitor, while its benzoylated counterpart BzLPRDSar showed the strongest effect against bacterial growth. Furthermore, it was shown that BzLPRDSar was able to prevent biofilm formation at concentrations that make it a potential lead compound. Computational data from our docking studies nicely explain this effect by the strongly increased hydrophobic interactions, when the N-terminus is benzoylated or carries a terminal Phe. Henceforth, experimental and computational data were use to build a preliminary pharmacophore model.We have synthesized a panel of 28 potential peptidomimetic SrtA inhibitors, of which several were shown to act as effective competitive inhibitors towards isolated SrtA. Among these, six compounds were found to have an ICi.e., S. aureus SrtA, with possible direct implementation in the prevention of septic arthritis. We believe that with optimization and in vivo testing, our peptidomimetic SrtA inhibitors could gain clinical potential, manifesting our strongest inhibitor as a potential lead. The inherent simple structure, non-toxicity of these peptides, combined with their high water solubility and low molecular weight makes them excellent starting points for drug development, in comparison to small organic natural compounds. Additionally, the inclusion of non-scissile bonds, such as sarcosine, in the most promising structure provides protection against host proteases, increasing the probability of the peptidomimetics to reach their targets. Furthermore, the potential of co-administering these SrtA inhibitors with either conventional antibiotics or compounds targeting other virulence factors could increase the clinical application of this strategy further. Taken together, with elucidation of exact structure–activity relationships and in vivo testing, antivirulent peptidomimetic SrtA inhibitors could be an excellent starting point to develop treatments counteracting the rapid increase of bacterial resistance to existing drugs.This work is a milestone towards the targeting of an essential virulence factor of Gram-positive pathogenic bacteria, Data will be made available upon request.in silico work.A. T. and D. T. designed the research and wrote the manuscript. H. A. and J. C. J. H. conducted the research and wrote the manuscript. J. C. J. H., S. R. and I. M. designed and conducted the bacterial experiments. D. T. conducted the There are no conflicts to declare.SC-014-D3SC01209C-s001