Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins , creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu. It is well known that the tumor microenvironment (TME) can induce antigen-specific tolerance or anergy by several different mechanisms . RestricTCR-engineered CD8 T cells have also been used in clinical trials , with efWhile co-stimulation can enhance TCR signaling and effector function as well as extend the operative life span of CTLs –28, T ceOur project aimed at generating a novel chimeric co-stimulatory switch protein (CSP) consisting of an intracellular co-stimulatory domains (ICD) fused to the extracellular domains (ECD) of a protein that utilizes ligands expressed in the tumor milieu. Such CSP should mediate antitumor effects on different levels including stromal attack and enhanced antitumoral T cell activity. CD40L (CD154) was selected as the donor protein for the ECD because of the pivotal role that the CD40/CD40L pathway plays in humoral and cellular immunity , 31. MorWe report here the molecular design of novel CD40L:CD28 CSPs and the functional activity of CD40L:CD28 CSP-engineered TCR-transgenic (tgTCR) T cells. Augmented T cell activity as well as DC maturation and induction of a pro-inflammatory secretome of tumor-conditioned DCs are demonstrated. CD40L:CD28 CSPs were observed to display a dynamic surface expression dependent on T cell activation and CD40 interaction. They delivered co-stimulatory signals and functional support to the T cells in a timely manner when the T cell interacted with the tumor cell and for as long as the tumor cell was present. Collectively, the concept of integrating T cell co-stimulation with the pathways that the CD40/CD40L interaction offers to support the immune response into a chimeric CD40L:CD28 design is a very promising approach to provide multiple key upgrades to T cells for enhanced efficacy in adoptive T cell therapy.Human PBMC from healthy donors were isolated by ficoll density gradient centrifugation and used for CSP transduction, isolation of B cells and monocytes. The blood collection was done with approval by the institutional review board of Ludwig-Maximilians-University, Munich, Germany. Donors gave informed consent in accordance with the Declaration of Helsinki. PBMC used for B cell isolation and DC generation were from HLA-A2 negative donors such that the B cells or DCs did not provide antigen ligands for the TCR-T58, TCR-D115 or TCR53- tgTCR-T cells in co-culture experiments.2 (PromoCell) for 24 h (Primary B cells were isolated from PBMC using the CD19 magnetic (negative isolation) Naïve B Cell Isolation Kit II, human , according to the manufacturer´s instruction. Primary B cells were used freshly prepared for the B cell activation assay. Monocytes were derived from PBMC by plastic adherence and were differentiated to immature dendritic (iDCs) by adding 100 ng/ml GM-CSF and 20 ng/ml IL-4  to VLE RPMI-1640 supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin and 5% human serum (DC-medium) on day 1 and day 3. iDC were obtained at day 7 and used for co-culture with T cells. Mature DC (mDC) were generated from day7 iDCs by adding Jonuleit maturation cocktail (20 ng/ml IL-4 (PromoCell), 100 ng/ml GM-CSF (PromoCell), 15 ng/ml IL-6 (Sigma-Aldrich), 10 ng/ml IL-1β (PromoCell), 20 ng/ml TNFα (Immunotools) and 1 µg/ml PGEfor 24 h .enriched in renal cell carcinoma) DCs as described , then centrifuged and filtered to remove cells -T cells either with HLA-A2 restricted specificity for the tyrosinase peptide (TCR-T58 or TCR-D115)  or with Cell lines were the human melanoma line SK-Mel23 , the human RCC cell lines RCC-53 and RCC-26 (isolated from RCC tissue in our laboratory) , the HEKThree different CD40L:CD28 CSP constructs were designed. Two of them created a type I membrane protein structure in which the C-terminal part corresponding to the soluble CD40L fragment (AA 113-261) was inverted and linked to the transmembrane (TMD) plus cytoplasmic domain (ICD) of CD28 (AA 153-220). For successful intracellular trafficking and surface expression, the PD-1 signal peptide (20 AA) was used. ECD and ICD were connected through a Glycine/Serine linker (10 AA), which provides flexibility of the connected functional domains, and a spacer, either IgG1Fc or Fil3 (third Ig-like repetition corresponding to the Filamin protein), for expression improvement. These CSPs were designated as CD40L:IgGFc:CD28 and CD40L:Fil3:CD28, respectively. The third construct adopted a type II transmembrane protein structure by linking the CD40L ECD and TMD (AA 14-261) with the inverted ICD (AA 180-220) of CD28. This construct was designated as CD40L:CD28i. The CD40L native sequence  and the chimeric sequences (ordered from GeneArt) were cloned in the pMP71 vector for retroviral transduction of primary T cells (CD3/CD28-activated PBMC), or tgTCR-T cells expressing TCR-T58, TCR-D115 or TCR53. Retroviral transduction was performed as described , 55. BriFor re-activation of T cells to induce CD40L:CD28 CSP surface expression, T cells, which were frozen on day 12 after CSP transduction, were thawed and stimulated either with anti-CD3 plus anti-CD28 antibodies or through target cell-expressed peptide/MHC TCR ligands.6 T cells/ml TCM with 100 U/ml IL-2). After 3 days, T cells were removed from the stimulation plate, diluted 1:4 and further cultured without anti-CD3/CD28 antibodies in TCM with 100 U/ml IL-2 for 3 additional days. On day 6, when endogenous CD40L was downregulated (evidenced by absence on CD4 Mock T cells by flow cytometry) and CSPs were still expressed, T cells were harvested and prepared for flow cytometry or used in B cell and DC activation assay.For anti-CD3/CD28 stimulation, CD40L:CD28 CSP-transduced T cells were thawed and seeded in 24-well non-tissue culture treated plates that had been coated with anti-CD3  and anti-CD28 antibodies (1 µg/ml)  before setting up the co-culture to block the endogenous CD40. After 24 h co-culture, cells were harvested for flow cytometry.To analyze surface expression of CD40L or CD40L:CD28 CSP, anti-CD40L-PE  was used in combination with anti-mouse TCRβ-constant region (mTCR)-PB  to detect the transgenic TCR expression, anti-CD4-APC-Cy7  and anti-CD8-V500 . 7-AAD (Sigma-Aldrich) was used for live/dead discrimination. HLA-A2 and CD40 expression on HEK293/Tyr cells, HEK393/Tyr/CD40 cells and tumor lines SK-Mel23, RCC-26 and RCC-53 were analyzed using anti-HLA-A2 (ATCC HB54) plus anti-mouse IgG1-A488 (Invitrogen) and anti-CD40-PE  together with 7-AAD. Flow cytometry was performed with the LSRII (BD) cytometer and FlowJo v10.7.1 software.2 to stimulate the T cells through the tgTCR-Tyr/HLA-A2 interaction and to trigger the CSPs through CD40. Unstimulated T cells were kept in culture as a negative control. After 30 min, co-cultures were immediately fixed using Cytofix Fixation Buffer (BD)  followed by permeabilization with ice cold Phosflow Perm Buffer III . Antibodies to surface markers, anti-CD45-PE-Cy7 , anti-CD3-PerCP-Cy5.5 , were added together with antibodies for the phosphorylated intracellular proteins p-AKT-PB , p-mTOR-A647  and p-RPS6-PE . Data were acquired on the LSRII cytometer and analyzed using the FlowJo v10.7.1 software.TCRtg-T cells transduced with CD40L:CD28 CSPs were thawed and activated for 6 days using anti-CD3 and anti-CD28 antibody coated plates to upregulate the surface expression of the CD40L:CD28 CSPs (see Re-Activation of T Cells). On day 6, when the endogenous CD40L was downregulated on T cells and CD40L:CD28 CSPs were still expressed, T cells were harvested and washed with PBS, then incubated for 4 h in RPMI-basic without IL-2 to reduce constitutive background p-AKT signals. After 4 h, T cells were harvested, washed with PBS/EDTA (2 mM), and stained with fixable Blue reagent  for viability (10 min), then washed and co-cultured with HEK293/Tyr/CD40 (1:2 ratio) for 30 min at 37°C and 5% CO2. Naïve B cells without stimulation were used as negative control. Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells. After overnight incubation, cultures were harvested and analyzed by flow cytometry for B cell specific surface activation markers CD83-PE , CD86-FITC  and Fas-PE-Cy7  together with CD19-A700  and 7-AAD.T cells transduced with CSPs and re-activated using anti-CD3 plus anti-CD28 antibody-coated 24-well plates (as described in Re-Activation of T Cells) were co-cultured at a 1:1 ratio with naïve human B cells per triplicate in 96-well U-bottom plate overnight at 37°C/5% CO6 cells) or ercDCs were harvested and co-cultured with T cells re-activated to express CD40L:CD28 CSPs (see Re-Activation of T Cells) at 1:1 ratios in 96-well U-bottom plates in a final volume of 200 µl TCM. Co-cultures were set in triplicates. iDCs alone, mDC, and iDCs with mock T cells were used as controls. After 24 h, supernatants were harvested for 45Plex bead array and cells were used for flow cytometry using antibodies to CD83-PE , CCR7-PB , PD-L1-PerCP-Cy5.5 , HLA-DR-APC-Cy7  and CD80-PE-Cy7  together with CD3-A700  and live/dead fixable blue stain .iDCs  with or without co-expression of the different CSPs were thawed and co-cultured without re-activation with SK-Mel23, RCC-26 or RCC-53 at 1:2 T cell to target cell ratio in a 96 wells. T cells and target cells cultured alone were used as controls. All target cells expressed CD40 endogenously. Supernatants were collected after 24 h and analyzed for IFN-γ secretion by ELISA.51chromium release assay as described  for 1 h at 37°C. TCRtg-T cells  with or without the co-expression of the different CSPs were thawed and plated without re-activation at titrated cell numbers reaching T cell to target cell ratios from 10:1 to 1.25:1 in a 96 well plate. Chromium-labelled targets cells were added at a concentration of 2000 cells per well. For spontaneous release, target cells were cultured without T cells. Each culture was set up in duplicates. Cells were co-cultured for 4 h at 37°C. For maximum 51chromium release, 50 µl of the target cell suspension was pipetted directly to the Luma plate . After the co-culture time, 50 µl aliquot from each co-culture well was transferred to the Luma filter plate, dried and counted using a TopCount machine. Specific cell lysis was calculated by applying the formula:T cell-mediated target cell killing was performed using a escribed . BrieflyIFN-γ analysis of T cell/tumor cell co-cultures was done using ELISA kits (BD Bioscience) according to the manufacturer´s instructions. Supernatants of T cell/DC co-cultures were analyzed using a multiplex bead array 45Plex  according to the manufacturer’s instructions. Data were acquired using the Luminex100 machine with BioPlex Manager 6.1 software (Bio-Rad Laboratories GmbH). Standard curves were fitted using the logistic-5PL regression type.According to the topological classification of transmembrane proteins, the CD28 protein and the CD40L protein belong to the type I and type II groups, respectively , 58. A tTo assess if the chimeric sequences are transcribed and translated correctly and are able to generate correctly folded proteins, which traffic to the cell surface, CSP-transduced T cells were stained with anti-CD40L antibody at different time points after transduction  to detect the CSP on the cell surface. T cells transduced with the native sequence of CD40L (nCD40L) were used as control. It was observed that the expression profile varied depending on the CSP design . The kinin vitro activation , B cells undergo activation, which can be detected by measuring key surface activation markers on the B cells like CD86, Fas and CD83  at a 1:1 ratio followed by analysis of surface activation markers on the B cell population by flow cytometry  were generated protocol , and theIn addition to effects on surface markers, CD40L:CD28-expressing T cells also caused changes in secreted cytokines and chemokines in co-cultures with DCs. These included augmented secretion of IL-12p70, IL-10, pro-inflammatory IL-1β, TNF, chemokines MIP-1α (CCL3), MIP-3α (CCL20) and MIP-3β (CCL19) as well as the cytotoxic protein granzyme B (gzmB)   , 69. erce ercDCs  for co-cIn a next step it was determined whether the CD28 ICD was capable to deliver a proper co-stimulation signal to the T cells. The CD28 co-stimulatory pathway starts with phosphatidylinositol 3′-kinase (PI3K) leading to phosphorylation of the protein Kinase B, also known as AKT, followed by downstream phosphorylation of the mammalian target of rapamycin (mTOR) molecule. Ribosomal protein S6 (RPS6) is then a downstream target of mTOR activation , 71.The functionality of the CD28 ICD within our CD40L:CD28 CSPs was tested by analyzing the phosphorylation of AKT, mTOR and RPS6 in CD40L:CD28 CSP-transduced tyrosinase-specific TCR-T58 T cells after stimulation with HEK293/Tyr/CD40 cells, which can trigger the T58-TCR through HLA-A2/Tyr ligands and the CD40L:CD28 CSPs through CD40 expression. Before setting up the stimulation co-culture, T cells were deprived of IL-2 for 4 hours to reduce the level of constitutive AKT activation. Subsequently, the starved T cells were co-cultured with the HEH293/Tyr/CD40 cells for 30 minutes, which was previously determined as the optimal time for AKT activation to reach its peak , 73.After 30 minutes of stimulation, phosphorylation of AKT protein was increased in CSP-transduced T cells in comparison with the Mock T cells without CSP or T cells carrying the native CD40L that does not contain the CD28 signaling domain, or the unstimulated T cells , while the MFI in Mock and native CD40L-expressing T cells was similar to unstimulated control  compared to TCR-T58/mock T cells without CD40L:CD28 CSP  had the lowest effect on cytokine secretion, while the CD40L:CD28i (purple) and the CD40L:Fil3:CD28 enhanced the cytokine secretion more strongly. This is of note, since it contrasts with the expression level of the CSPs on the T cells where the CD40L:IgGFc:CD28 consistently had highest levels and CD40L:Fil3:CD28 was lowest  and type I (CD28) membrane protein. The principles of this type of combination are not yet clearly defined  and reprvia spacer and Glycine/Serine linker, as commonly used in the CAR design  was inverted and connected to the transmembrane plus intracellular CD28 signaling sequence R design , 75. TwoR design . The FilR design , conferrFor the third CD40L:CD28 design, the extracellular and transmembrane domains of CD40L were kept in the orientation of the native CD40L protein and were directly linked to the inverted intracellular co-stimulatory domain of the CD28 protein (CD28i). The final structure of the third CSP CD40L:CD28i resembled a type II membrane protein and had a similar size to the endogenous CD40L protein. This approach offered a design with less structural modifications and suggested a more natural interaction with the CD40 receptor.After retroviral transduction, the CD28i and the IgGFc constructs were highest expressed, while the Fil3 construct was in comparison poorly expressed. Although retroviral transduction is a process that should result in stable expression of transduced sequences, this was not the case for the CD40L:CD28 CSPs. Over 13 days of culture, expression of all constructs gradually decreased. The Fil3 CSP was almost undetectable after 6 days, the nCD40L was lost at day 10, the CD28i CSP at day 13, and the IgG1Fc CSP remained detectable by day 13 at 52% expression compared to the initial level.Culture conditions, T cell activation using anti-CD3 and anti-CD28, and specific activation signals delivered by TCR-MHC/peptide interaction were able to induce re-expression of the CSP on T cells and the presence of the CD40 receptor on the target cells caused its subsequent downmodulation.It is known that endogenous CD40L expression on CD4 T cells exhibits a continuous cycle of downregulation and re-expression upon interaction with CD40 expressing B cells, with this being dependent on the presence of antigen , 76, 77.in vivo circumstances. A beneficial co-stimulatory effect might, thereby, occur timely coupled with the recognition of tumor cells through the TCR-MHC interaction, providing help for improved T cell functionality for as long as tumor cells are present and the antitumoral response is required. The “inbuilt” stopp of the T cell support after removal of the activator (i.e. antigen-positive tumor or infected cell) could prevent undesired over-activation of the immune system through the CD40L domain of the CSP.In our study, similar to the one described above , the CD4In terms of biological activity, the extracellular CD40L domain of the CSPs was able to functionally interact with B cells and DCs making them potentially more effective antigen presenting cells. The extent of effect correlated to the expression level on the T cell surface with the IgGFc construct showing highest effects followed by the CD28i CSP and Fil3 CSP. Changes included the upregulation of MHC class II and co-stimulatory molecules CD80/CD86) as well as maturation markers, like CD83, CCR7 and PD-L1 as was expected from the literature , 80, 81./CD86 as Moreover, T cells that were equipped with CD40:CD28 CSPs stimulated iDCs to secrete pivotal cytokines and chemokines for lymphocyte recruitment and T cell stimulation. These included IL-12p70, pro-inflammatory IL-1β, and TNF, as well as MIP-3α (CCL20) and MIP-3β (CCL19), which are chemotactic for lymphocytes and DCs, and IFN-α, which is known to activate antigen presentation for T cell-mediated tumor cell recognition . NotablyCD40L:CD28-transgenic T cells induced similar effects on ercDCs, which are tumor-conditioned myeloid cells found in human RCC , 69. ErcCollectively, the observed effects on B cells and DCs confirm the functionality of the CD40L extracelluar domain and, moreover, provide support for our proposed hypothetical mode of action of CD40L:CD28 CSP-expressing T cells where tumor-resident APCs can receive the benefit of stimulation through the CSP to rescue their activity in the TME.For the CD28 domain, functionality was demonstrated by augmented phosphorylation of the AKT protein and downstream AKT targets, mTOR and RPS6 proteins, after co-culturing the CSP-transduced T cells with antigen-specific CD40-positive target cells. The strength of the effect was inversely associated with the level of CSP surface expression on T cells: The most highly expressed IgGFc CSP evoked weakest activation of the phosphorylation cascade while the Fil3 CSP, although being low expressed, and CD28i CSP (despite its inverted orientation) had stronger effects.The presence of the CD28 signaling domain in the CSP conferred improved cytokine secretion and antigen-specific cytotoxicity to tgTCR T cells when in contact with target cells expressing specific antigens and the CD40 receptor. The extent to which T cell function was enhanced correlated to the level of AKT pathway activation which was best achieved through the CD40L:Fil3 and CD40L:CD28i CSPs. High surface expression was apparently not determining the signaling quality and cis-effect on T cell functionality. Explanations for this observation remain speculative. The CD40L-CSP containing the IgGFc spacer is the longest one of the three CD40L:CD28 CSPs. The longer distance of the extracellular domain from the membrane surface might impact the signaling outcome as has been seen in CAR designs , 89. TheCD40 agonists are explored in clinical trials aiming at moderating macrophages and stroma influences as well as augmenting response to chemotherapy and immunotherapy –92. ConcThe results are part of a doctoral thesis submitted to the Ludwig Maximilian University of Munich.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ludwig Maximilians University, Ethical Committee. The patients/participants provided their written informed consent to participate in this study.Conception and design, LO-C, AM, BH, GP and EN. Development of methodology, LO-C, AM, and EN. Acquisition of data, LO-C. Analysis and interpretation of data, LO-C, AM, and EN. Writing of the manuscript, LO-C and EN. Review, and/or revision of the manuscript, LO-C, AM, GP, BH, and EN. All authors contributed to the article and approved the submitted version.The work was supported through funding Deutsche Krebshilfe, SFB-TR36, Erich & Gertrud Roggenbuck Stiftung and DAAD-CONACyT scholarship cooperation program.EN declares financial relationship due to patent WO2017/162797.The remaining author(s) declare(s) that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The pathology of Alzheimer’s disease (AD), the most common cause of dementia, is considered to be mainly driven by two major hallmarks (tau and amyloid beta). It is highly desirable to develop an affordable medicinal diagnostic that can be utilized worldwide for the early diagnosis of AD. Hence, p-tau231 was selected as a specific target, which appears both in AD serum and cerebrospinal fluid, for the development of a sensing platform for the diagnosis of AD. To the best of our knowledge, these are the first aptamer-mediated biosensors that rely on sensitive fluorescent and colorimetric aptasensors for the rapid monitoring of p-tau231. The nitrogen-doped carbon dot-based turn-on fluorescent aptasensor could rapidly analyze p-tau231 down to 3.64 ng/mL within 40 min, and the colorimetric Cu-enhanced-Au aptablot displayed high sensitivity at 4.71 pg/mL through a digital camera, with visibility to the naked eye down to 8 ng/mL p-tau231 within 140 min. Owing to their advantages, which include affordability, rapidity, high sensitivity, and dependence on complicated instruments, these aptamer-based biosensors offer significant potential for the early diagnosis of AD worldwide. Alzheimer’s disease (AD) is the most common form of neurodegenerative dementia that severely endangers the elderly, both physically and mentally. The disease is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brain; the former is due to the aggregation of amyloid β peptide (Aβ), while the latter is formed by the polymerization of phosphorylated tau protein ,2,3. AmoThe nanobiosensor development for AD diagnosis has been well investigated during the last decade, with a variety of techniques, such as ELISA, colorimetric assay, and electrochemical assays ,9,10. HoIn the present work, we have developed two promising aptasensors for the detection of p-tau231 as AD biomarkers, including a fluorescent aptasensor and a colorimetric aptablot, for the detection of p-tau231, using fluorescent nitrogen-doped CDs (NCDs), gold nanoparticle (AuNPs), and specific aptamer of p-tau231. 6H8O7·H2O) and sodium citrate dihydrate (C6H5Na3O7·2H2O) were purchased from OCI Company . Polyethylenimine  and sodium ascorbate were obtained from Alfa Aesar . Tetrachloroauric(III) acid (HAuCl4·3H2O) was purchased from Sigma–Aldrich . Nitrocellulose membrane was purchased from Nupore, India. Human serum albumin (HSA) was also received from Sigma–Aldrich . Phosphate buffer saline  was purchased from Tech-Innovation . p-tau231, amyloid beta 1–42 peptide (Aβ42), C-reactive protein (CRP), tumor necrosis factor α (TNF-α), and insulin were obtained from Abcam . Amyloid beta 1–42 aggregates containing Aβ42 oligomer were obtained from Anaspec . Thiol-modified AFB1 aptamer (5′-HS-(CH2)6-CAGCACCGTCAACTGAATGGGGAGAGTGGTGGGGCGGGGGCCGGATCCGTGATGCGATGGAGATGT-3′) was synthesized by Genotech Corp. , according to identification from the literature [All reagents were of analytical grade and were used for the experiment without further purification. Deionized (DI) water at 18.2 MΩ cm was purified using a Milli-Q system . Citric acid monohydrate  water, and air-dried. NCDs were successfully synthesized using a hydrothermal method. Citric acid monohydrate  and PEI1800  were dissolved in 30 mL DI water, and vigorously stirred for 30 min at room temperature (RT). The mixture solution was placed in a Teflon-lined autoclave reactor, and heated at 190 °C for 1 h. A short reaction time (1 h) was chosen to fabricate the NCDs to produce NCDs with high quantum yield . NCDs we® X-ray diffractometer . UV-Vis and fluorescent spectra of NCDs were acquired by multi-mode microplate reader .The physical properties of the as-prepared NCDs were obtained through characterization. The size of NCDs was obtained by high-resolution transmission electron microscopy . The structures of the different components of the NCDs were analyzed through X-ray photoelectron spectroscopy (XPS) spectra using X-ray photoelectron spectrometry . X-ray diffraction (XRD) data were measured on SmartLab2+ (1 mM) and different concentrations of p-tau231 contained in PBS (180 µL) were introduced to the NCDs/aptamer complex solution, and incubated for different incubation time at 37 °C. Finally, the fluorescence intensities of the reaction mixture were continuously measured under 365 nm excitation. The fluorescence recovery after the addition of p-tau231 was evaluated by calculating the intensity ratio (F’-F)/F, where F is the fluorescence emission intensity after fluorescence quenching by aptamer, and F’ is the recovery fluorescence intensity after adding p-tau231. To monitor the interference effects of different biomarkers that appear in human serum, such as human serum albumin (HSA), amyloid beta 42 monomer (Ab42), amyloid beta 42 oligomer (AbO42), C-reactive protein (CRP), tumor necrosis factor α (TNF-α), and insulin; 2.5-fold concentration of these biomarkers was used to measure the interference effect to turn-on the fluorescent aptasensor for p-tau231.The p-tau231 aptamer solution  was incubated at 90 °C for 5 min, and gradually cooled to RT for 15 min. One hundred microliters of p-tau231 aptamer solution was mixed with NCDs in 1.5 mL microcentrifuge tube to quench the fluorescence of NCDs. The mixture solution was thoroughly pipetted, and equilibrated for different incubation times (0–50) min at 25 and 37 °C. The final concentration of Mg2, PEI1800, and sodium ascorbate) to improve the color intensity [Gold nanoparticles were synthesized by the reduction of sodium citrate, following the literature . The aptntensity . These cNCDs were successfully synthesized using citric acid and PEI1800 as carbon source and nitrogen source, respectively, through a one-step hydrothermal method. The morphology and size of NCDs confirmed by TEM show the spherical shape of the as-synthesized NCDs with estimated size of 3.5–4.5 nm, and the zeta potential of +27.0 mV indicates the high dispersibility and colloidal stability of the NCDs . 2+ ions, Mg2+ ions  could accelerate the saturated folding transition of aptamers into the specific shape that selectively bind to their targets [p-tau231 is considered as a promising biomarker of the emerging AD pathology because p-tau231 is neuropathologically increased earlier than p-tau181, not only prior to the positive threshold of amyloid beta, but in response to early brain tau deposition ,7,22. Th targets ,26, lead2+ ions that were added along with p-tau231, according to the above interference study of ferrous ions detection. However, after 15 min, the fluorescence recovery was gradually observed, and reached equilibrium recovery within 40 min incubation time. The 40 min incubation time after the addition of p-tau231 was chosen for further experiments. 2 = 0.995) in the range 0–75 ng/mL  in the p-tau231 concentration range of 0–1000 ng/mL  and the colorimetric AuNPs-based aptablot , respectively. Although these approaches displayed high specificity towards other AD biomarkers, further characterization is required to assess the specificity of these approaches towards other phosphoepitopes. These proposed approaches exhibit many obvious advantages that include multifunctionality, low cost, simple and rapid sensing process, high sensitivity and specificity, and independence from complicated instruments and technical professionals. These aptamer-based biosensors for p-tau231 might significantly pave the way for diagnostic biosensors towards the early diagnosis of AD worldwide.


More important, the TRBV6-5-TRBD2-TRBJ2-7 combination with high frequency was shared between CD4+ T and CD8+ T cells of different COVID-19 patients. Finally, we found the dominant characteristic motifs of the CDR3 sequence between recovered COVID-19 and healthy control. Our study provides novel insights on TCR in COVID-19 with different convalescent phases, contributing to our understanding of the immune response induced by SARS-CoV-2.Coronavirus disease 2019 (COVID-19) is a global infectious disease caused by the SARS-CoV-2 coronavirus. T cells play an essential role in the body’s fighting against the virus invasion, and the T cell receptor (TCR) is crucial in T cell-mediated virus recognition and clearance. However, little has been known about the features of T cell response in convalescent COVID-19 patients. In this study, using 5′RACE technology and PacBio sequencing, we analyzed the TCR repertoire of COVID-19 patients after recovery for 2 weeks and 6 months compared with the healthy donors. The TCR clustering and CDR3 annotation were exploited to discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. We first identified CD4 COVID-19 is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which usually leads to respiratory infections, and severe cases develop into severe pneumonia or even death [Beginning in December 2019, the coronavirus disease 2019 (COVID-19) outbreak has posed a serious threat to more than 200 countries worldwide and has caused more than 5.2 million deaths  on the cell surface using hypervariable T cell receptor (TCR). TCR diversity is widely recognized as a direct measure of immune competence, as it quantifies the variety of foreign antigens and hence acts upon them. At the early stage of T cell development, TCR is generated by somatic rearrangement of variable (V), diversity (D), and joining (J) gene segments, known as V(D)J recombination . Thus, t+ and CD8+ T cell activation and interferon type I and III responses. The emergence of shared T cell clusters occurs in the rehabilitation of COVID-19 patients by next-generation sequencing technology [+ T cells [+ T cell in convalescent COVID-19 patients by single-cell sequencing [Recently, sequencing technology has been widely used to reveal activation-induced phenotypic profiles of antigen-reactive T cells ,21. Somechnology . It has  T cells . Notablyquencing . Additioquencing . However+ and CD8+ T cells of convalescent patients after SARS-CoV-2 infection, and then further analyzed the characteristics of their TCR repertoire of convalescent patients with different convalescent stages.In this study, we used the 5′RACE (Rapid Amplification of cDNA Ends) method combined with third-generation high-throughput sequencing technology to investigate the TCRα and TCRβ library sequences on peripheral blood CD4Nine confirmed COVID-19 patients of 2 weeks convalescence and five healthy controls were obtained from Fifth Medical Center of Chinese PLA General Hospital, Beijing, China. The clinicopathologic characteristics of the study group participants with 2 weeks convalescence and healthy controls are listed in + T and CD8+ T cells were purified from PBMCs with the Easysep® human CD4+ or CD8+ positive selection kits from Stem Cell technologies . Purified cells were eluted and washed with PBS containing 2% (v/v) fetal bovine serum (FBS) and 1mM/L EDTA.PBMC were isolated with the Ficoll-Paque density gradient centrifugation protocol. The CD4+ T and CD8+ T cells, total RNA was extracted using RaPure Total RNA Micro Kit  according to the manufacturer’s instructions. A universal primer binding site, sample barcode, and unique molecular identifier (UMI) sequences were introduced using the SMARTer® RACE 5′/3′ kit  with TCRα and TCRβ constant segment-specific primers  for cDNA synthesis. cDNA libraries were amplified in two PCR steps, introducing the second sample barcode and Illumina TruSeq adapter sequences at the second PCR step. The PCR program for both rounds was: 5 cycles at 94 °C for 30 s, 5 cycles at 68 °C for 30 s, and 25 cycles at 72 °C for 3 min (first-round PCR) and 40 cycles at 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 2 min (second-round PCR). The PCR products were separated on 1.5% agarose gel by electrophoresis.For the sorted CD4We measured the concentration of PCR products with a barcode and mixed the same amount of PCR products from all COVID-19 patients and healthy controls. The mixed PCR products were separated with 2% agarose gel, and then the extracted DNA was sent to Novogene Institute . The amplicons were sequenced on a PacBio Sequel system using V3 chemistry . The raw data (raw reads) in FASTQ format were first processed using Python and custom Perl scripts. Clean data (clean reads) were obtained after removing various reads, including poly-N reads, reads without a 3′ adapter or an insert tag, reads with 5′ adapter contamination, reads with poly-A, T, G, or C low-quality reads from the raw data. Moreover, the Q20, Q30, and GC contents of the raw data were calculated. All sequences shorter than 200 bps, having homopolymers of 6 bps barcodes, containing primer mismatches, and with a quality score lower than 19, were removed.http://www.r-project.org, accessed on 10 November 2021).The processing stage of TCR repertoire analysis started with the mapping of V, D, and J segments. Filtered reads were subsequently aligned to V, D, J, and C gene segments of TCR alpha (TRA) and TCR beta (TRB) locus for clonotype assembly of complementarity-determining region 3 (CDR3) nucleic acid sequences by IMGT/HighV-QUEST web server. Furthermore, the rarefaction curve of the TCR repertoire was generated using VDJtools for each subject. We obtained the core tabular format for VDJ tools by the conversion supported in the program, which contained count, frequency, CDR3 nucleotide sequence, Vend, D start, D end, and J start. The frequency of various V–J junctions was calculated and displayed using basic analysis of VDJtools, making circus-style V–J usage plots. Additionally, the diversity estimation was utilized to compute a set of diversity statistics as well as to visualize the repertoire clonality and the comparison of diversity estimates. The V(D)J patterns of each sample were combined to analyze the dominant rearrangement pattern in different groups. The CDR3nt and CDR3aa in advantageous V(D)J usage were merged to further search for the dominant CDR3. All statistical analysis was implemented with R software  and TCRβ (TRB) chains of antigen-specific T cells . For each blood sample, we purified CD4 T cells . A total T cells .+ T cells compared with CD4+ T cells. At the same time, no significant difference was observed in overall frequencies of abundant TRA and TRB clonotypes in convalescent COVID-19 patients, relative to healthy controls .To further explore whether a unique V(D)J recombination pattern is specific for convalescent COVID patients, we next compared the V(D)J paring of TRA and TRB in each individual separately. In the CD47/TRAJ54 C, and 23 T cells D. Similapatients E,F. Of npatients D,F, sugg antigen D,F. Addi+ T cells and CD8+ T cells of convalescent COVID-19 patients. Therefore, we identified the dominant CDR3 sequences in each group. Due to the highly diverse nature of CDR3, only a few consensus sequences were identified, of which two CDR3 sequences of TRA were shared in CD8+ T cells of two-week convalescent COVID-19 patients  the changes in TCR clones during the different convalescent phases of COVID-19 and the priority use of V and J gene fragments in COVID-19 patients compared with healthy controls; (b) there is persistent TCR dominant V(D)J recombination during the convalescent phase of COVID-19 from 2 weeks to 6 months; (c) the unique CDR3 motif specific for SARS-CoV-2 in CD8In recent years, TCR repertoire in various diseases was characterized using TCR-seq. Multiplex PCR-, target enrichment-, and 5′ RACE (RNA only)-based approaches are the most widely used TCR-seq strategies. When using multiplex PCR, preferential amplification of highly abundant gene products can occur, contributing to inaccuracies in reported clone frequencies. Additionally, intronic sequences and incomplete VDJ recombination products may contribute to data noise when using gDNA samples. Target enrichment strategies reduce the extent of amplification bias seen in standard multiplex PCR but are more labor/time-intensive and do not resolve the issues associated with gDNA input . However+ T cells and CD8+ T cells. Although CD4+ T and CD8+ T cells exhibit their roles differently, CD8+ T cells recognize and kill virus-infected cells through TCR-mediated viral antigens [+ T cells have multiple roles in coordinating and mediating immune responses against viruses [+ T and CD8+ T cells can participate in the joint resistance to SARS-CoV-2 infection via the production of a virus-specific TCR recombination pattern.The TCR is composed of TCRα and TCRβ chains, which containing VJ and VDJ gene segments, separately . The resantigens , while C viruses . Therefo+ T but not CD4+ T cells of convalescent COVID-19 patients. Moreover, no identical CDR3 motifs have not been reported relative to the published SARS-CoV-2-related TCR clusters. Our results suggest that these CD8+ T cells with unique SARS-CoV-2-associated CDR3 motifs might play a crucial role in the immune response to eliminate the SARS-CoV-2 virus efficiently. However, these unique T cell sequence motifs require prospective validation to be used in COVID-19 patients, and specific T cells reactive to SARS-CoV-2 epitopes also need to be identified in the future. Therefore, our TCR data might help to fingerprint a shared clonal expression T cell phenotype that existed within a population of COVID-19 patients and that defined anti- SARS-CoV-2 immunity in individuals.In the process of V(D)J recombination, random nucleotide deletions and insertions often occur in the CDR3, which is essential for antigen binding . Therefo+ T and CD8+ T cells, such as TRBV5-4/D2/TRBJ2-5 and TRBV5-4/D1/TRBJ2-2, appear at the 2-week convalescent stage and continue to the 6-month convalescent stage, supporting that these T cell clones might have long-term memory characteristics after SARS-CoV-2 infection to participate in the elimination of residual SARS-CoV-2 in the convalescence stage.Furthermore, T cells with long-lasting memory have been detected in patients recovered from SARS-CoV-2 infection , and SAROur findings reveal that COVID-19 patients have unique cellular immune characteristics during the different convalescent phases, which contributes to our understanding of the immune response induced by SARS-CoV-2 and might be used as a basis for prognostic evaluation and targeted therapy for COVID-19 patients.


Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.Agonists of the G Chronic pain is a highly debilitating condition, disturbing all aspects of our daily experience in social and career-related contexts. The pharmacological tools currently available are sometimes inadequate, or, as in the case of opioids, limited by serious adverse effects . Thus, e1, A2A, A2B and A3 receptors . A1Rs and A3Rs are coupled to Gi members of the G protein family, while A2ARs and A2BRs are Gs-coupled receptors adenosine (DPMA), nor the A3R agonist, 2-hexyn-1-yl-N6-methyladenosine (HEMADO), had any effect on glutamate-induced pain. The authors conclude that peripheral A1Rs alleviate, while peripheral A2AR exacerbate, acute nociception, whereas A2BRs and A3Rs do not participate in this particular pain mechanism amino]-9H-purine-9-yl]-N-methyl-β-D-ribofuranuronamide) and its chlorinated counterpart Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide), is not associated with cardiac or hemodynamic effects [3R-selective nucleosides are already in phase 2 and/or 3 clinical trials for autoimmune inflammatory diseases, liver cancer, and non-alcoholic steatohepatitis  [In contrast, the activation of the A effects , thus po&rank=1) . Hence, 3R is Gi-coupled, similarly to the A1R, and its activation decreases intracellular cAMP levels, an effect classically related to pain control [3R-mediated effects on pain were described, it was found that the local application of the A3R agonist produced an intrinsic, formalin-like, nociceptive response, and potentiated the algesic effect of low formalin concentrations [3R activation was later ascribed to the stimulation of this receptor subtype on mast cells [3R activation does not induce degranulation and histamine release in human mast cells [The A control . Howevertrations ,45. The st cells , where ist cells . Howeverst cells .3R activation in different rodent models of neuropathic pain. Works performed by the team of Salvemini demonstrated that a single, intraperitoneal (i.p.) injection (at day 7 after injury) of prototypical  or newly synthesized (MRS1898) A3R agonists proved effective in relieving neuropathic pain caused by CCI or chemotherapy treatment in rodents [3R antagonist MRS1523, but not to the A1R and A2AR blockers DPCPX and SCH-442416, respectively, nor to the opioid antagonist naloxone [3R-mediated pain control was achieved after a single dose of IB-MECA administered at the peak of CCI-induced allodynia (on day 7 after nerve ligation: D7), and was not different from that produced by consecutive daily injections of the compound (from D8 to D15). Thus, it was shown that efficient A3R-mediated pain relief is achieved after a single acute administration of the agonist, and that the A3R does not become tolerant to agonist activation [3R agonist treatment did not affect the physiological pain threshold in the contralateral paw, indicating a specific anti-hyperalgesic effect in conditions of altered sensitivity that causes the selective alleviation of persistent neuropathic pain states, but without analgesic effects [3R agonist-mediated pain relief was absent in A3R knock-out (KO) mice [More recently, extensive evidence has highlighted the opposite, i.e., anti-hyperalgesic, effect of A rodents . These enaloxone . Of notetivation . It is w effects ,50. A3R 3Rs was further confirmed in mice and rats using a variety of pain models [3R ligands, including innovative, highly selective A3R agonists synthesized by Jacobson and coworkers  . So, in  MRS1523 . Again,  MRS1523 . Similar MRS1523 . This me MRS1523  by astro MRS1523 .3R-mediated pain control is efficiently achieved in mice and rats [3R agonists. All effects were prevented by the spinal administration of the selective A3R antagonist MRS1523 [3R-mediated anti-hyperalgesia are still not fully understood and, as stated below, peripheral routes of action have also been described.Taken together, these data indicate that Aand rats , and is and rats ,51,52,53and rats ,58,59,60and rats  or spinaand rats ,50,51) a3R-induced pain relief. Janes et al. (2014) demonstrated that A3R agonists block the development of chemotherapy-induced neuropathic pain (CINP) by exerting beneficial effects associated with the modulation of spinal neuro-glial communication and neuroinflammatory processes. IB-MECA administered i.p. 15 min prior to each paclitaxel dose attenuated astrocytic activation in the spinal cord by inhibiting NADPH oxidase, and by enhancing redox-sensitive nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and MAPK kinases such as ERK1/2 and p38. This results in a reduced level of the neuroexcitatory/proinflammatory cytokines TNF-α and interleukin-1β (IL-1β), whereas an increase in the neuroprotective/anti-inflammatory interleukin-10 (IL-10) was observed, as well as restored synaptic glutamate homeostasis at the dorsal horns [3R, as later data demonstrate that microglial activation was not observed during the development of oxaliplatin-induced mechano-hypersensitivity in rats [3R-mediated inhibition of redox state alterations in the spinal cord during chemotherapy treatment results in a second A3R-mediated protective mechanism, e.g., the maintenance of glutamate homeostasis at the synapse, because IB-MECA prevented post-transcriptional alterations in glutamate transporter-1 and glutamine synthetase [Various molecular mechanisms have been proposed to mediate Aal horns   receptor antagonist bicuculline [+-Cl− co-transporter 2- (KCC2)-maintained Cl− gradient [3R agonists (IB-MECA and MRS5698) prevented GAT-1 and GAD65 inactivation, and promoted Cl− gradient maintenance by preserving KCC2 functionality, thus counteracting extracellular GABA decrease and preventing mechanical allodynia  applied to isolated DRG neurons decreased neuronal firing and inhibited pro-nociceptive Ca2+ currents sensitive to the N-type channel blocker PD732121, an analogue of ω-conotoxin [1Rs by CPA led to a much smaller Ca2+ current (ICa) inhibition than Cl-IB-MECA, and the effect of the endogenous agonist adenosine was blocked to a higher extent by the A3R-selective antagonist VUF5574 than by the A1R-selective blocker DPCPX. Adenosine-mediated ICa inhibition was completely abolished by a combination of both agonists, demonstrating that A1R and A3R, but not the A2R subtypes, concurrently inhibit N-type ICa (Cav2.2) in rat DRG neurons, but that A3R-mediated effects predominate [2+ channels (VDCCs) in isolated mouse DRG neurons [1R activation [3R activation inhibits Ca2+ entry into the neurons and action potential firing, suggesting an inhibition of synaptic transmission at the dorsal horn. However, direct evidence for decreased glutamate release into the spinal cord is still lacking. These results are also in line with previous evidence demonstrating that the aberrant expression and/or activity of N-type VDCCs is associated with neuropathic pain [2+ channel block, likely due to their wide expression in the CNS.Beyond above mentioned effects on the CNS, further evidence from Coppi et al. (2019) shows that peripheral mechanisms could be involved in Aonotoxin   treatment [3R agonist MRS5980, at the peak of visceral hypersensitivity (day 14 after DNBS injection) was found to be effective in relieving visceral allodynia measured by quantifying the number of abdominal muscle contractions upon intestine distention [2+ channel blocker PD723212 mimicked A3R agonist-mediated pain control, thus confirming previous evidence implicating this ion channel in DRG electrical activity [A similar mechanism of action, e.g., Cav2.2 inhibition, has been proposed by Lucarini et al. for Areatment . In thisstention . Interesactivity   mice infiltrated the inflamed DRG and restored A3R agonist-mediated anti-allodynia [+ T cells from A3R-KO or IL-10-KO mice did not restore the A3R-mediated anti-hyperalgesic effect, demonstrating, for the first time, that A3R activation on CD4+ T cells elicits IL-10 release, which, in turn, is responsible for the anti-hyperalgesic effect of MRS5980 [3R-mediated anti-allodynic effect were elucidated in the same work by use of an innovative in vitro model consisting of co-cultures of DRG neurons and T cells isolated from the same animal, either naïve or CCI mice. By this paradigm, CD4+ or CD8+ T cell infiltration into the ganglion was reproduced in vitro via a circumscribed experimental model, wherein any eventual effects of the cytokine(s) released by the T cells on DRG neurons upon A3R stimulation could be evaluated in isolation. Notably, the application of the A3R agonist MRS5980 to DRG-CD4+ T cell co-cultures significantly reduced the action potential (AP) firing evoked by a depolarizing current ramp and increased the current threshold for AP initiation. The effect was not observed in DRG-CD8+ T cell co-cultures, or in DRG neurons cultured alone, demonstrating the selective engagement of A3Rs expressed on CD4+ T cells [+ T cells upon A3R activation, inhibiting neuronal excitability  [3R activation might facilitate the balance between pro-inflammatory and anti-inflammatory pathways in favor of the latter.Interestingly, a recent paper outlined that the pain-relief properties of some tricyclic antidepressants, such as amitriptyline, one of the most widely prescribed antidepressants for neuropathic pain management , might bn (CREB) . This su3R-mediated pain control has recently been outlined. Indeed, differences in the effects of A3R agonists in male and female rodents with paclitaxel-, oxaliplatin- or bortezomib-induced peripheral neuropathy have been reported. The study shows that the A3R agonist MRS5698 failed to prevent bortezomib-induced neuropathic pain in female, but not male, rodents [3R ligands. In the same work, the authors suggested that the lack of effect of MRS5980 in female rats was likely due to the absence of A3R overexpression in the spinal cord after bortezomib treatment, in contrast to male rats, in which A3R is overexpressed [Interestingly, a certain degree of sexual dimorphism in A rodents . Of notexpressed .3R agonists could be attractive co-therapeutics with opioids. Indeed, the prevention of morphine-induced AK overexpression in the dorsal horn of the spinal cord provided a >90% attenuation of antinociceptive tolerance in CCI mice, an effect mimicked by the selective A3R agonists IB-MECA and MRS5698 [3R stimulation in mechanical allodynia were associated with the inhibition of the NOD-like receptor pyrin domain-containing 3 inflammasome in the spinal cord, leading to decreased IL-1β production and increased IL-10 levels [3R activation following treatment with levels of A3R agonist (i.th.) that had no effect on nociceptive threshold in morphine-naïve rats potentiated/restored antinociception (tail flick assay) in morphine-tolerant rats [Finally, A MRS5698 . Interes0 levels . Furtherant rats .3Rs induces potent anti-hypersensitive effects in diverse preclinical models of chronic pain. Nevertheless, the mechanism by which this AR subtype exerts anti-hyperalgesic and anti-allodynic effects is still to be clarified, and before now, both peripheral  and central  effects have been described, with some gender-specific effects outlined in particular cases . Of note, the efficacy of A3R ligands for the pharmacological control of chronic pain states is particularly relevant because it is devoid of tolerance effects  or significant side effects . However, the clinical relevance of A3R-mediated pain control still needs to be demonstrated, and, even if results from clinical trials for other pathologies encourage the use of these ligands for their safe profile, future work will help us elucidate the feasibility of A3R-based treatments in humans.The modulation of A


Background: National Institute on Aging—Alzheimer's Association (NIA-AA) proposed the AT(N) system based on β-amyloid deposition, pathologic tau, and neurodegeneration, which considered the definition of Alzheimer's disease (AD) as a biological construct. However, the associations between different AT(N) combinations and cognitive progression have been poorly explored systematically. The aim of this study is to compare different AT(N) combinations using recognized biomarkers within the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort.Methods: A total of 341 participants were classified into cognitively unimpaired  and cognitively impaired  groups according to the clinical manifestations and neuropsychological tests. Cerebrospinal fluid (CSF) Aβ42 and amyloid-PET ([18F]flutemetamol) were used as biomarkers for A; CSF phosphorylated tau (p-tau) and tau-PET ([18F]flortaucipir) were used as biomarkers for T; CSF total tau (t-tau), hippocampal volume, temporal cortical thickness, [18F]fluorodeoxyglucose (FDG) PET, and plasma neurofilament light (NfL) were used as biomarkers for (N). Binary biomarkers were obtained from the Youden index and publicly available cutoffs. Prevalence of AT(N) categories was compared between different biomarkers within the group using related independent sample non-parametric test. The relationship between AT(N) combinations and 12-year longitudinal cognition was assessed using linear mixed-effects modeling.Results: Among the CU participants, A–T–(N)– was most common. More T+ were detected using p-tau than tau PET (p < 0.05), and more (N)+ were observed using fluid biomarkers (p < 0.001). A+T+(N)+ was more common in the CI group. Tau PET combined with cortical thickness best predicted cognitive changes in the CI group and MRI predicted changes in the CU group.Conclusions: These findings suggest that optimal AT(N) combinations to determine longitudinal cognition differ by cognitive status. Different biomarkers within a specific component for defining AT(N) cannot be used identically. Furthermore, different strategies for discontinuous biomarkers will be an important area for future studies. Alzheimer's disease (AD) is the most common cause of dementia, and one of the main causes of complications and death in the aging population. A series of complex pathobiological processes is involved in the pathogenesis of AD, including the deposition of extracellular amyloid plaques, tau-related intracellular neurofibrillary tangles (NFTs), neuronal loss and atrophy .All participants in this study were from the Alzheimer's Disease Neuroimaging Initiative (ADNI), a longitudinal multicenter study designed to develop clinical, imaging, genetic, and biospecimen biomarkers for tracking the progression of AD. Regional ethics committees of all institutions approved the ADNI study. Written informed consent was obtained from all participants. The key eligibility criteria were based on the ADNI protocol. Cognitively unimpaired (CU) participants must be free of memory complaints and cognitively normal, with MMSE scores between 24 and 30 (inclusive) and a CDR of 0. Cognitively impaired (CI) participants must have a subjective memory concern and were divided into two subgroups, namely: mild cognitively impaired (MCI) participants and AD dementia participants. The MCI participants reported MMSE scores between 24 and 30 (inclusive), a memory complaint, have objective memory loss measured by education-adjusted scores on Wechsler memory scale logical memory II, a CDR of 0.5, absence of significant levels of impairment in other cognitive domains, essentially preserved activities of daily living, and an absence of dementia. The AD dementia participants met NINCDS/ADRDA criteria for probable AD , phospho-tau (181P), and total tau were analyzed using the electrochemiluminescence immunoassays (ECLIA) Elecsys following a Roche Study Protocol  vs. ICV (x) within 128 ApoE-negative CU participants  PET, respectively. The [18F]florbetapir standardized uptake value ratios (SUVRs) were calculated by averaging the four cortical regions, frontal, which are anterior/posterior cingulate, lateral parietal, and lateral temporal cortices  and Clinical Dementia Rating Sum of Boxes (CDRSB). According to the interquartile range , we selected seven-time points from baseline to 12 years  for the longitudinal cognitive assessment.AT(N) biomarkers included CSF Aβ42 (A1), amyloid PET ([18F]florbetapir) (A2), CSF p-tau (T1), tau PET ([18F]flortaucipir) SUVR in the ITC (T2) and Braak V/VI region (T3), hippocampal volume ([N]1), temporal meta-ROI cortical thickness ([N]2), CSF t-tau ([N]3), AD-characteristic FDG PET SUVR ([N]4), and plasma NfL ([N]5). For CSF Aβ positivity, we used a published cutoff , Cohen's kappa coefficient (κ), and percentage agreement (concordance). Prevalence estimates for AT(N) categories were calculated with 95% confidence intervals generated using bootstrap resampling . Prevalence of AT(N) categories was compared between different biomarkers within the group using related independent sample non-parametric test (McNemar test for A and Cochran's Q-test for T or [N]). The relationships between AT(N) combinations and cognitive trajectories  were examined using a linear mixed-effects (LME) model  with subject-specific intercepts and slopes. The goodness of LME models with different AT(N) combinations was assessed by marginal R2, which represented the fixed effect of LME models. All analyses were performed using IBM SPSS Statistics 20, with significance of the two-tailed test set to p < 0.05.Demographics and continuous biomarkers were compared between different groups using Mann–Whitney p < 0.01) . Therefore, the prevalence of (N)+ using NfL was likely to vary by age in the present cohort, so we calculated the cutoff based on age stratification.Demographics are presented in 3 (N1), temporal meta-ROI thickness <2.9214 mm (N2), CSF t-tau > 233.6 ng/L (N3), FDG PET meta-ROIs <1.2599 SUVR (N4), and plasma NfL levels >30.35 ng/L in younger participants and >36.45 ng/L in older participants. Similar cutoffs were obtained using 90% sensitivity for AD, while mean ± 2 SD from Aβ-negative CU controls resulted in more conservative cutoffs (Cutoffs were defined as CSF Aβ42 <880 ng/L (A1), amyloid PET >1.1 SUVR (A2), p-tau > 21.11 ng/L (T1), ITC tau PET >2.122 SUVR (T2), Braak V/VI tau PET >1.938 SUVR (T3), adjusted hippocampal volume < -0.4477 cm cutoffs .p < 0.05) and vs. NfL . The highest positivity for T was observed when CSF p-tau were used for both A+ or A– (p < 0.001)  categories in CU and CI participants is summarized in < 0.001) . Based oN) biomarkers, the most prevalent category was A-T-(N)- (range 26.1%  to 50.8% ). Although eight possible categories were identified for each AT(N) variants, A+T+(N)+, A+T-(N)+, and A-T+(N)+ had very low frequencies when using MRI imaging and FDG PET. A+T+(N)-, A+T-(N)+, and A-T+(N)- were almost lacking in the combination of CSF p-tau and t-tau since a strong correlation  and almost perfect agreement  was observed between them, as previous study reported  (When adding (< 0.001) .p < 0.05) and lower prevalence of T+ using tau PET (p < 0.005)] (p < 0.001). In participants with MCI, A-T- were the most common categories when using tau PET in Braak V/VI (p > 0.05) . A and T categories of different AT(N) variants in the CI group showed similar trends to the CU  . There waak V/VI . In the  > 0.05) .p > 0.05) (When adding (N) biomarkers, the most prevalent category was A+T+(N)+ (range 29.9%  to 51.8% ), and the frequencies of T+(N)- and T-(N)+ in the combination of CSF p-tau and t-tau were relatively low . As ment > 0.05) .p = 0.027 and education, p = 0.048 in CDRSB; age, p = 0.025 and education, p < 0.001 in MMSE), consistent with previous findings   . After considering the random effect, the conditional R2 increased to 19.32% and 33.55% for the CDRSB and MMSE scores, respectively. These results indicated that longitudinal cognition in CU participants was mainly associated with individual characteristics, and MRI measurements were the best biomarkers to predict cognitive changes.The overall findings for longitudinal cognition using continuous predictors are summarized in in MMSE) . The besin MMSE) . We consp = 0.061) and t-tau (p = 0.051) in CDRSB, and the marginal R2 using MRI imaging and tau PET was relatively higher than others  (t-test (p < 0.001 for CDRSB and MMSE), and interactions dominated the main effects. Again, CSF Aβ42, tau PET, and temporal cortical thickness were the best combinations in both scales, and all interactions were significant   biomarkers could predict longitudinal cognition, except CSF p-tau (n others . The AT( 35.57%) . Then, w 50.25%) .Finally, similar findings were observed when using the LME model with time as a covariate to verify the results using continuous predictors .We repeated the AT(N) prevalence analyses using alternative cutoffs . Using cIn this study, we found that different combinations of AT(N) biomarkers exerted different effects on the category prevalence and predictions of cognitive decline. First, the difference in the composition of AT(N) categories between CU and CI individuals is not surprising. Categories representing the AD continuum were the most common in CI participants, while more subjects with non-AD pathological changes were observed in the CU group  prevalence, we noted that both AT(N) categories and variants differed between CU and CI participants. Normal AD biomarkers (A-T-[N]-) and non-AD pathological change  account for most CU individuals, whereas the Alzheimer's continuum  accounts for CI individuals, especially AD   categories and found that the results were not completely consistent. This finding highlights that the optimization of categorization strategies is important for future studies.R2 was obtained when using CSF t-tau to predict longitudinal cognition in CI participants. Firstly, CSF t-tau was reported to be related to multiple variables , which may attenuate the association with cognition under adjustment for such covariables  variants on longitudinal cognition evaluated using both the CDRSB and MMSE. CDRSB may enable a more detailed analysis of subtle changes with different stages of dementia severity  framework provides a more precise division of the Alzheimer's continuum based on the pathology  study. The patients/participants provided their written informed consent to participate in this study.R-RL: analysis and interpretation of the data and drafting the manuscript. Y-YX, X-YL, and Y-HC: data acquisition, analysis, and interpretation of the data. Q-QT: funding, design of the study, and critical revision of the manuscript. Z-YW: funding, conceptualization and design of the study and critical revision of the manuscript. All authors reviewed the manuscript, contributed to the manuscript revising and editing critically for important intellectual content, given final approval of the version, agreed to be accountable for all aspects of the work presented here, and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, RS1A, was stabilized against proteolysis by all five cathepsins  while inhibiting Cts L with >25-fold specificity against the other cathepsins. We further showed that this stabilized RS1A peptide could inhibit Cts L in human liver carcinoma lysates (IC50 = 19 μM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation  Information on the effects of sidechain and backbone modification on the activity of cathepsin (Cts) L, V, K, S, and B was used to design a thioamide peptide that is inert to all Cts and selectively inhibits Cts L. Additionally, the ease of obtaining high biological and chemical diversity from standard synthetic procedures makes peptide therapeutics attractive.1,4 Peptides thus stand out as promising candidates to fill the gap between the two main drug categories – traditional small-molecule drugs  and biologics (larger than 5000 Da).4 Most of the peptides that are currently clinically approved or under active development are targeted for metabolic diseases and cancer.2 However, despite the numbers of known targets for peptide therapeutics and existing peptide libraries, peptides still display certain disadvantages that hinder them from more easily becoming effective drugs.1,2 Peptides are subject to rapid proteolysis, oxidation, display short half-lives and fast renal clearance in vivo, as well as low membrane permeability, thereby exhibiting suboptimal pharmacokinetics.1,2 To address the metabolic stability issues of peptides, modifications at protease cleavage sites using techniques such as synthetic substitutions of amino acid sidechains or the peptide backbone have been developed and utilized to increase resistance to proteolysis.3,5–8In recent decades, there has been an increased interest in the development of peptides as therapeutics and imaging agents.9–20 Our laboratory previously demonstrated that a thioamide substitution near the scissile bond of glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP), two therapeutically relevant peptides for diabetes treatment, significantly enhances their proteolytic stability against dipeptidyl peptidase 4.21 Thioamidation of GLP-1 and GIP increased their half-lives up to 750-fold without significantly compromising their cellular activity; the thioamide GLP-1 analogue was also biologically active in rats and exhibited improved potency for glycemic control compared to its native, all-amide GLP-1 counterpart.21 Motivated by these results showing thioamide stabilization effects at P2 and P1 positions (positions numbered from the scissile bond by convention), our laboratory developed a fluorescence sensor design to systematically study the positional effects of thioamide substitution against different cysteine proteases  and serine proteases .22–24 Intriguingly, we found that thioamide positional effects differ not only between serine proteases and cysteine proteases, but also between members of the same protease family despite their high homology (31–59% sequence identity) and mechanistic similarity.22,24 We also successfully utilized data from these systematic studies to design a two-site stabilized thioamide peptide specifically targeting neuropeptide Y1-receptor expressing MCF-7 breast cancer cells.22 With the experimental data from these systematic studies, we recently developed a Rosetta machine learning model that accurately classifies positional effects of thioamides on proteolysis by these cysteine and serine proteases which can be used to rationally design stabilized peptides for therapeutic and imaging applications.23Backbone thioamidation is a promising tool that has been shown to improve proteolytic stability of both linear and macrocyclic peptides.25,26 Cysteine Cts proteases, which comprise 11 members in humans , belong to the papain-like cysteine protease family. They have been shown to be upregulated in many cancer types and play critical roles in cancer progression.27,28 Cts L is an ubiquitously expressed endopeptidase that is uniquely involved in the major histocompatibility complex (MHC) class II processing pathway,29 prohormone or proneuropeptide processing,30–32 and autophagy,33 as well as cardiac homeostasis and signal transduction.34–37 Cts L is highly expressed in tumors associated with breast cancer, colorectal cancer, and pancreatic adenocarcinoma.27,38 Cts L participates in the degradation of epithelial cadherins, transmembrane receptors, and extracellular domains of cell adhesion molecules in cancer cells, thereby disrupting cell adhesion, promoting tumor invasion, and possibly underlying resistance to chemotherapy.27,39,40 Importantly, Ou et al. recently showed that lysosomal activation of SARS-CoV-2's spike (S) glycoproteins by the host cell's Cts L, but not Cts B, is critical for its cellular entry via endocytosis during infection.41 These researchers showed that treatment with Cts L inhibitor SID 26681509 decreased SARS-CoV-2 pseudovirus entry into HEK 293/hACE2 cells by more than 76%, highlighting the role of Cts L in lysosomal priming of the virus upon entry.41 There is also evidence for elevated Cts L circulating level in COVID-19 patients.42 This is significant as Cts L inhibitors have now been identified as promising therapeutic agents to inhibit SARS-CoV-2 for potential treatment of COVID-19.41–44 It has been proposed that a protease inhibitor cocktail composed of a Cts L-specific inhibitor as well as serine protease inhibitors could be a safe and novel treatment for COVID-19 patients.45 Although it is desirable to develop Cts L inhibitors, there are currently no specific inhibitors for Cts L that have advanced to clinical trials as it is challenging to obtain selectivity against closely related Cts family members.46Given this precedent, in this study, we aim to further utilize the strategic incorporation of thioamides to develop stabilized peptides as protease inhibitors, more specifically, inhibitors of the cysteine protease cathepsin L (Cts L). Among the 500–600 proteases identified in mouse and human, the cathepsin (Cts) family includes proteases that orchestrate numerous critical physiological processes and are involved in many different diseases such as neurological disorders, cardiovascular diseases, arthritis, obesity, and cancer.e.g. epoxide ring, acyloxymethyl ketone, aziridine, vinylsufonate, nitrile, or thiosemicarbazone) that are strategically placed to trap the catalytic Cys25 residue of Cts L.46 This follows a logic common to the development of covalent protease inhibitors, wherein a good protease substrate is converted into an inhibitor by strategic incorporation of such warheads.46–48 Inspired by this principle, we demonstrate herein that good peptidyl substrates of Cts L, designed by combining knowledge about substrate sequence specificity from previous positional scanning with our protease sensor studies, can be converted into good inhibitors of Cts L by a single thioamide substitution to the peptide backbone. Unlike the warhead strategy, thioamide modification only renders the substrate inert to proteolysis and does not result in covalent inhibition. There are many advantages to our strategy as concerns about the use of covalent enzyme inhibitors linger in spite of several successes with the aforementioned warhead approach.46,49,50 With our thioamidation approach, we hope to potentially overcome challenges with selectivity and off-target effects of Cts inhibitors that are normally encountered with small molecule protease inhibitors.46 An inhibitor with high specificity for a single Cts is a powerful tool compound for studying its role in health and disease and can serve as a therapeutic lead where such specificity is necessary to avoid undesirable side effects. In this study, we examine the stability of several thioamide peptide scaffolds toward Cts proteolysis and identify one peptide that shows resistance to Cts L, Cts V, Cts K, Cts S, and Cts B while inhibiting only Cts L. We also show that this stabilized thioamide peptide can inhibit Cts L in human hepatocellular liver carcinoma (HepG2) whole cell lysate. To our knowledge, this is the highest affinity thioamide-based protease inhibitor to date. Our studies show the potential of utilizing thioamides to stabilize and convert good peptidyl substrates into specific protease inhibitors.Many of the known Cts L inhibitors, which are mostly small molecules, resemble its physiological substrate and often have electrophilic “warheads”  at both the N-terminus and C-terminus to systematically study thioamide positional effects on proteolysis of cysteine24 and serine22 protease substrates. Once the doubly-labeled peptide is cleaved, there will be a turn on in fluorescence as one of the fluorophores will be separated from the thioamide, regardless of the placement of the thioamide.22,24 This allows real-time monitoring of proteolysis kinetics. From our previous systematic studies with cysteine proteases , we learned that thioamide substitution at the P1 position significantly slowed the proteolysis rates of the generic μLLKAAAμ substrate by Cts V, Cts K, Cts S, and Cts L, but not significantly by Cts B and papain.23,24 By convention, amino acids N-terminal to the scissile bond are denoted PX positions , while those C-terminal to the scissile bond are considered PX′ positions . Interestingly, we found that the P1 thioamide peptide, μLLKSAAAμ (KSP1), not only showed the highest level of protease resistance to Cts L, but also served as a potent inhibitor of Cts L 23,24 as the sequence of this peptide was designed to be generic. Motivated by these results, we thus envisioned advancing this approach to design and scan for a sequence-optimized, thioamide-containing peptide specific inhibitor to Cts L, yet being stabilized in the presence of other closely related cathepsins without inhibiting them.Previously, our laboratory has designed a fluorescent protease sensor system that capitalizes on the fluorophore quenching property of thioamides to monitor real-time protease activity.via steady-state protease assays  at this position.53,54 Similar preference for aromatic residues at P2 position is only observed in Cts V, which is most closely related to Cts L by sequence identity (78% sequence identity).53 As Cts V favors tryptophan and tyrosine over phenylalanine, phenylalanine seemed to be the best choice for P2. For P4, Cts L shows a preference for histidine, prompting us to choose histidine at this position. At P3, however, Cts L has less well defined specificity, but displays some preference for basic residues as well as a few aliphatic amino acids.53 Since we already included a positively charged residue at the P1 position, we decided to incorporate an aliphatic amino acid at P3 to reduce the potential for multiple Cts L cleavage sites. We chose leucine for the P3 position since the data from Choe et al. suggested that Cts L prefers leucine at P3 among the aliphatic amino acids , μHLFKAAAμ (K3A), and their P1 thioamide analogs (RS3A and KS3A)  to design all-amide peptides that are good substrates of Cts L, then (2) to turn those substrates into stabilized peptides inhibiting Cts L by strategic placement of a single thioamide. In this study, utilizing the peptide sensor design from our previous studies, our peptide inhibitor candidates contained two coumarins (μ residues) at their termini, allowing for quick initial identification of peptides that showed resistance to proteolysis by cathepsins e assays .22,24 To. Fig. S1.53 With s Fig. S1.53 Regarnd KS3A) . The thi3A and R3A were recognized and efficiently cleaved by all five proteases, confirming that these were indeed good substrates of Cts L  was recognized and cleaved by all five cathepsins – Cts L, Cts V, Cts K, Cts S, and Cts B  as discussed in the next section.53To validate our design, we needed to first confirm whether the all-amide peptides were good substrates of the proteases before proving that the thioamide substitution could transform them into stabilized peptide inhibitors. For ease of comparison between thioamide positions and protease, raw fluorescence measurements were normalized and are presented in of Cts L . It is w Table S5. Interesby Cts L . The onlAμ RS1A; , would end Cts B , S10 whit intact , S11. Si3A and R3A peptides and their P1 thiopeptides) as well as the RS1A peptide , allowing them to be separately monitored without interference. The all-amide peptides (K3A and R3A) showed some inhibitory effects, which was expected since these substrates compete for the active site of Cts L , which showed resistance to proteolysis by all five cathepsins, was also a good inhibitor of Cts L with a KI value of 1.11 ± 0.22 μM. Although the RS3A and KS3A exerted slightly better inhibitory effects than the truncated peptide RS1A, they are not as ideal because we established in the steady-state protease assays that they could be cleaved by Cts L and by Cts B at the C-terminus. Lastly, the role of the two coumarins was examined with the coumarin-free peptide HLFRSA (RS1A*). Although this peptide showed resistance to cleavage by Cts L , indicating an important role for the coumarins in binding. The finding that the thioamide peptides could serve as potent inhibitors of Cts L was exciting because our previous investigations of thioamide-stabilized protease substrates had found them to be only fairly weak inhibitors, implying that the thioamide primarily acted to disrupt binding to the protease.21,22,24 Indeed, earlier investigations of thioamide effects on proteolysis had found similar results for di- and tripeptides.14,15,20,55 Thus, we wished to further investigate the mechanism of inhibition.Inhibition assays with Cts L were performed with all of the peptides from the first series  using GraphPad Prism software.56 All of the α values are consistently between 1–2, confirming these peptides are likely mixed-type inhibitors, with element of competitive inhibition since a value of α > 1 suggests tighter binding to the free enzyme  with a mixed inhibition mechanism that can inhibit Cts L-mediated cleavage of the viral glycoproteins derived from all four viruses – SARS-CoV, Ebola, Hendra, and Nipah viruses, a process that is essential for entry into host cells.58From initial evaluation of the kinetic parameters obtained by fitting data to a Michaelis–Menten model  by Cts K or Cts S in the presence of >30 μM concentrations of RS1A peptide  substrate in the presence of up to 50 μM RS1A peptide with Cts B , the RS1A peptide is ≥25-fold selective against other Cts family members tested, as it only weakly inhibits the closely related Cts V (78% sequence identity with Cts L) and shows little to no significant inhibitory effects with Cts K, Cts S, and Cts B .To serve as useful specific inhibitors of Cts L, the thioamide peptides must also be inert to cleavage by other proteases that may be present 38,61 In particular, it has been previously shown that increased Cts L expression is associated with worse outcome in hepatocellular carcinoma patients62 and elevated Cts L activity has been found in malignant liver cancer HepG2 cells.63 To further validate our RS1A peptide inhibitor of Cts L, we investigated whether it could effectively inhibit Cts L activity in whole cell lysate from the HepG2 human hepatocellular liver carcinoma cell line. Using a commercially available fluorescence based Cts L activity kit, we incubated different doses of RS1A peptide with HepG2 whole cell lysate. We found that the RS1A peptide could effectively inhibit fluorescent reporter activity in the HepG2 whole cell lysate (IC50 = 19.3 ± 4.5 μM)  . Signifil lysate  and S26†S3A and the truncated peptide RS1A with Cts L and the other four cathepsins investigated in this project. Interestingly, exclusively in the Cts L simulations, we observe that the P1 thioamide bond N–H of the RS3A peptide can interact with His163, which is part of the Cts L catalytic triad , which is consistent with the fact that Cts B is both an endopeptidase and a carboxydipeptidase.28 Upon examination of the docked structure of the RS3A and Cts B, we found that the carboxylic acid of the C-terminal μ of the peptide interacts with His112 on the occluding loop, which is one of the two histidines (His111/His112 or His110/His111) known in the literature to anchor the C-terminal carboxylate of substrates to give Cts B its carboxydipeptidase properties  for the two Cts L peptides of interest , but is also a potent, specific inhibitor of Cts L. This peptide can reversibly inhibit Cts L without degradation in HepG2 liver cancer cell lysate and shows promising activity in MDA-MB-231 breast cancer cells. Such a peptide is desirable since peptide-based agents, especially those targeting proteases, are often subject to degradation in vivo. Furthermore, reversible inhibitors like this could potentially address the safety concerns from lack of specificity and potential elicitation of immune responses with irreversible, covalent inhibitors.49,50 While the selectivity against other cathepsins is not as high as some previously reported peptidomimetics ,45 this has not been our focus here. Rather, we sought to demonstrate that one can rationally design a potent reversible protease inhibitor by strategic modification of amino acid sidechains and thioamide position based on sensor data from our own work and others. More detailed mechanistic studies, as well as further optimization of this peptide for higher affinity and selectivity will be pursued and reported subsequently. Our studies show the potential of utilizing thioamides as stabilized peptide inhibitors and reaffirm the value of thioamides in the peptide drug design toolbox. In future studies, we will further optimize the thioamide peptide scaffolds by exploring substitutions with unnatural amino acids as well as more carefully examining the role of the N- and C-terminal coumarin groups, removal of which led to a 13-fold decrease in KI. More rigorous biological studies, including assessment of cell permeability, are also warranted to more fully assess the utility of these compounds for in vivo studies of Cts L and possible therapeutic advancement. Given our previous success in machine learning approach to predict thioamide effects and the existing database of sequence effects on cathepsin activity, we may be able to computationally design peptide-based inhibitors for cathepsins as well as for other targets.23 Taken together these approaches can form a paradigm for developing thioamide-stabilized peptides as enzyme inhibitors.In summary, we have examined several thioamide peptide scaffolds and identified one peptide, RFor a typical trial, a 7.5 μM peptide solution was incubated in the absence or presence of the appropriate concentration of Cts L (30.3 nM), Cts V (20.5 nM), Cts K (42.6 nM), Cts S (21.6 nM), or Cts B (37.6 nM) in 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, and pH 5.5 buffer at 27 °C. The fluorescence was monitored as a function of time at 390 nm with an excitation wavelength of 325 nm using a Tecan M1000 plate reader. Three independent trials were conducted for each assay to ensure reproducibility. More details of the assays, along with the raw data and analysis, are included in the ESI.†For Cts L, various concentrations of its substrate Z-Phe-Arg-AMC  were reacted with 37.93 nM Cts L. For Cts V, different concentrations of its substrate Z-Leu-Arg-AMC  were reacted with 19.3 nM Cts V. For Cts K, different concentrations of the substrate Z-LR-AMC  were reacted with 53.2 nM Cts K. For Cts S, various concentrations of the substrate Z-LR-AMC  were reacted with 33.8 nM Cts S. Lastly, for Cts B, various concentrations of the substrate Z-Arg-Arg-AMC  were reacted with 40.9 nM Cts B. All assays were performed in an assay buffer consisted of 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, and pH 5.5 in a 96-well plate at 27 °C. The peptide inhibitors were pre-incubated with the appropriate proteases in the assay buffer for 10 min to ensure full interactions prior being added to the fluorogenic substrates. The fluorescence of the reaction was monitored as a function of time at 460 nm with an excitation wavelength of 380 nm by a Tecan M1000 plate reader. Each assay was done in triplicates to ensure reproducibility. Details of the analysis for these assays are outlined in the ESI.†−1; ab166833; Abcam, Cambridge, MA) was evaluated using a fluorometric Cathepsin L Activity Assay Kit  following the manufacturer's protocols. Briefly, in each well of a 96-well plate, 50 μL of the HepG2 cell lysate diluted in CL buffer  was incubated with 50 μL of CL buffer without (Control) or with the presence of different peptide inhibitor RS1A concentrations . The cell lysate and the peptide inhibitor were incubated at room temperature for 10 min. A total of 2 μL of 10 mM CL substrate Ac-FR-AFC substrate  was then added to each well, except the Lysate Background Control wells. Different concentrations  of SID 26681509, a known Cts L inhibitor, were used as positive controls. The samples were mixed; the plate was sealed to avoid evaporation and incubated at 37 °C for 1 h. The fluorescence of each sample was measured at 505 nm with an excitation wavelength of 400 nm on the Tecan plate reader. More details of the assay and data fitting are included in the ESI.†Cts L activity in human hepatocellular carcinoma HepG2 whole cell lysate (200 μg at 2.5 mg mL1BP4) which contains a peptide-like covalent inhibitor68 was used as a template in order to provide a reasonable starting structure for docking. Manual docking was performed by replacing the native covalent inhibitor with the WHLFRAAAW peptide which was prepared using PyRosetta.69 The cathepsin proteases of interest, Cts B (PDB ID 1GMY),70 Cts K (PDB ID 1BGO),71 Cts L (PDB ID 3HHA),72 Cts S (PDB ID 1MS6),73 and Cts V (PDB ID 1FH0),74 were aligned to the manually docked papain complex using PyMOL. The cathepsin protease WHLFRAAAW starting complexes were formally docked by performing the FlexPepDock protocol in Rosetta in order to optimize the binding interaction between the proteases and peptides of interest.75 The tryptophan residues in WHLFRAAAW were mutated to 7-methoxycoumarinyl alanine (μ) residues using the MutateResidue tool in PyRosetta toolbox with a params and rotamer library generated previously.23 Next, a constrained FastRelax was performed in PyRosetta in order to accommodate the newly mutated 7-methoxycoumarinyl alanine residues. A flat harmonic constraint was used to maintain proximity of the scissile bond to the active site cysteine residue. Thioamides were introduced into the relaxed complexes through patches written previously.23,76 The thioamide containing peptides were then simulated with five independent local relax trajectories without any constraints.In order to simulate the protease/peptide complexes from this study, the structure of the papain protease  between the residue total energies  of the three residues of the protease catalytic triad as well as the P1 and P1′ residues of the peptide were computed from all of our FlexPepDock models. These five energy score deltas were reduced into three dimensions with Principal Component Analysis.77 The three principal component axes were then clustered with the KMeans algorithm utilizing four clusters which was derived by maximizing the Silhouette heuristic.78 The three-dimensional data were plotted and visualized with matplotlib.79Unsupervised machine learning was performed by clustering energy features from PyRosetta modeling with scikit-learn.Kinetic and inhibition data with associated fitting as well as structural models of the peptides in complex with proteases are available as ESI. A key file is provided with descriptions of each data file.H. A. T. P. and E. J. P. designed the experiments. H. A. T. P. performed all of the experiments with some guidance and preliminary inhibition data from T. M. B. and C. L. S. G. G. performed computational modeling and machine learning analysis. H. A. T. P. and E. J. P. performed the experimental data analysis and structural model analysis. H. A. T. P. and E. J. P. wrote the manuscript with input from all other authors.There are no conflicts to declare.SC-012-D1SC00785H-s001SC-012-D1SC00785H-s002SC-012-D1SC00785H-s003SC-012-D1SC00785H-s004SC-012-D1SC00785H-s005SC-012-D1SC00785H-s006SC-012-D1SC00785H-s007SC-012-D1SC00785H-s008SC-012-D1SC00785H-s009SC-012-D1SC00785H-s010SC-012-D1SC00785H-s011SC-012-D1SC00785H-s012SC-012-D1SC00785H-s013SC-012-D1SC00785H-s014SC-012-D1SC00785H-s015SC-012-D1SC00785H-s016SC-012-D1SC00785H-s017SC-012-D1SC00785H-s018SC-012-D1SC00785H-s019SC-012-D1SC00785H-s020SC-012-D1SC00785H-s021SC-012-D1SC00785H-s022SC-012-D1SC00785H-s023SC-012-D1SC00785H-s024SC-012-D1SC00785H-s025SC-012-D1SC00785H-s026SC-012-D1SC00785H-s027SC-012-D1SC00785H-s028SC-012-D1SC00785H-s029SC-012-D1SC00785H-s030SC-012-D1SC00785H-s031SC-012-D1SC00785H-s032SC-012-D1SC00785H-s033SC-012-D1SC00785H-s034