Human Cysteine Proteinase Research Articles

N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 μM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.

High-throughput modular click chemistry synthesis of catechol derivatives as covalent inhibitors of SARS-CoV-2 3CLpro

The 3C-like protease (3CLpro) is a crucial target in anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) drug design. Herein, we performed high-throughput synthesis of catechol derivatives from the bioactive catechol-terminal alkyne scaffold A4, by using modular click chemistry. Subsequently, we conducted two rounds of SARS-CoV-2 3CLpro inhibition screening and selected seven compounds for synthesis and further efficacy validation. Compound P1-E11 had potent inhibitory effects toward SARS-CoV-2 3CLpro (IC50 = 2.54 ± 0.46 μM); exhibited good selectivity toward the human cysteine proteases cathepsins B and L; and demonstrated superior anti-SARS-CoV-2 potency (EC50 = 4.66 ± 0.58 μM) with low cytotoxicity (CC50 > 100 μM) in A549-hACE2-TMPRSS2 cells. The irreversible covalent mechanism of P1-E11 was confirmed through time-dependent experiments, enzyme kinetic studies, and dilution and dialysis assays. The binding affinity between P1-E11 and SARS-CoV-2 3CLpro with a KD value of 0.57 μM was validated through surface plasmon resonance (SPR) experiments. Molecular docking provided insights into the binding mode of P1-E11 to the target protein. This study demonstrated the feasibility and efficacy of modular click reactions in natural-product-based structural modifications and presents a novel approach for leveraging this strategy in antiviral drug discovery.

The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism.

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule's lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug's nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.

A comprehensive review on human disease-causing bacterial proteases and their impeding agents.

Proteases are enzymes that catalyze the amide bond dissociation in polypeptide and protein peptide units. They are categorized into seven families and are responsible for a wide spectrum of human ailments, such as various types of cancers, skin infections, urinary tract infections etc. Specifically, the bacterial proteases cause a huge impact in the disease progression. Extracellular bacterial proteases break down the host defense proteins, while intracellular proteases are essential for pathogens virulence. Due to its involvement in disease pathogenesis and virulence, bacterial proteases are considered to be potential drug targets. Several studies have reported potential bacterial protease inhibitors in both Gram-positive and Gram-negative disease causing pathogens. In this study, we have comprehensively reviewed about the various human disease-causing cysteine, metallo, and serine bacterial proteases as well as their potential inhibitors.

The Diagnosis Significance of Serum Cysteine Protease Inhibitors (CST4) in Colorectal Cancer.

This study aims to evaluate the diagnostic value of human serum cysteine protease inhibitors (cystatin 4 [CST4]) in colorectal cancer (CRC) patients. A total of 291 patients who were admitted to Zhuzhou Central Hospital for colonoscopy from January 2020 to December 2021 and met the inclusion criteria were selected. Serum samples of the patients were collected, and CST4 was detected by double-antibody sandwich enzyme-linked immunosorbent assay. Simultaneously, CEA and CA19-9 were detected, and the patients were divided into the CRC group, benign lesion group, and healthy control group. An attempt was made to construct a CRC prediction model including CST4 and draw a subject working characteristic curve as a diagnostic threshold for CRC prediction, and evaluate the diagnostic efficacy of the above indicators. At the same time, the expression analysis of CST4, CEA, and CA19-9 was verified by combining the data of CRC in the Tumor Genome Atlas (TCGA). In this study, the levels of serum CST4, CEA, and CA19-9 in the CRC group were higher than those in the colorectal benign lesion group and healthy control group, with statistical significance (P < .001). The analysis results of the receiver operating characteristic curve showed that the area under the receiver operator characteristic curve (AUC) of CST4 was 0.7739, which was obviously larger than the AUC of CA19-9 and CEA. CRC data from the TCGA expression database showed that CST4 expression and CEA expression were higher in CRC patients than in normal samples. The combined model based on CST4 was successfully constructed, and the AUC for predicting the occurrence of CRC was 0.7851. CST4 is a novel and improved diagnostic marker for CRC. The combined model based on CST4 has a certain potential value in terms of predicting the occurrence of intestinal cancer.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 μM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 μM), similar to that of remdesivir (EC50 = 2.27 μM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID‐19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID‐19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain‐like protease), and recombinant human cathepsin L. Celastrol suppressed SARS‐CoV‐2 replication in human and monkey cell lines and decreased interleukin‐6 (IL‐6) secretion in the SARS‐CoV‐2‐infected human cell line. Celastrol acted in a concentration‐dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS‐CoV‐2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID‐19 due to its ability to suppress SARS‐CoV‐2 replication and IL‐6 production in infected cells.

The Remarkable Selectivity of Nirmatrelvir

The SARS-CoV-2 main protease is among the most attractive targets for the development of therapeutic interventions for COVID-19. Successful candidate agents will not only possess potent on-target activity versus SARS-CoV-2 Mpro but also minimal polypharmacology versus human cysteine proteases. This Viewpoint explores the activity profile of the first approved SARS-CoV-2 Mpro inhibitor (Nirmatrelvir) versus a panel of cysteine proteases and considers the therapeutic implications of the data.

Expression signature, prognosis value and immune characteristics of cathepsin F in non-small cell lung cancer identified by bioinformatics assessment

BackgroundIn recent years, immunotherapies and targeted therapies contribute to population-level improvement in NSCLC cancer-specific survival, however, the two novel therapeutic options have mainly benefit patients containing mutated driven genes. Thus, to explore other potential genes related with immunity or targeted therapies may provide novel options to improve survival of lung cancer patients without mutated driven genes. CTSF is unique in human cysteine proteinases. Presently, CTSF has been detected in several cell lines of lung cancer, but its role in progression and prognosis of lung cancer remains unclear.MethodsCTSF expression and clinical datasets of lung cancer patients were obtained from GTEx, TIMER, CCLE, THPA, and TCGA, respectively. Association of CTSF expression with clinicopathological parameters and prognosis of lung cancer patients was analyzed using UALCAN and Kaplan–Meier Plotter, respectively. LinkedOmics were used to analyze correlation between CTSF and CTSF co-expressed genes. Protein–protein interaction and gene–gene interaction were analyzed using STRING and GeneMANIA, respectively. Association of CTSF with molecular markers of immune cells and immunomodulators was analyzed with Immunedeconv and TISIDB, respectively.ResultsCTSF expression was currently only available for patients with NSCLC. Compared to normal tissues, CTSF was downregulated in NSCLC samples and high expressed CTSF was correlated with favorable prognosis of NSCLC. Additionally, CTSF expression was correlated with that of immune cell molecular markers and immunomodulators both in LUAD and LUSC. Noticeably, high expression of CTSF-related CTLA-4 was found to be associated with better OS of LUAD patients. Increased expression of CTSF-related LAG-3 was related with poor prognosis of LUAD patients while there was no association between CTSF-related PD-1/PD-L1 and prognosis of LUAD patients. Moreover, increased expression of CTSF-related CD27 was related with poor prognosis of LUAD patients while favorable prognosis of LUSC patients.ConclusionsCTSF might play an anti-tumor effect via regulating immune response of NSCLC.

The Effect of Dipeptidyl Nitrile Derivatives on Pancreatic Ductal Adenocarcinoma Cells In Vitro

Aims: This study aims to evaluate the bioactivity of dipeptidyl nitrile inhibitors of human cysteine cathepsins that could work as anticancer agents in a drug discovery and development project. Background: Human lysosomal cysteine proteases promote cancer progression, migration, and metastasis, targeted by inhibitors. Objective: Here, 19 cysteine protease inhibitors known as dipeptidyl nitriles were tested using MIA PaCa-2 pancreatic cancer cells and Balb/3T3 clone A31 non-tumoral mouse fibroblasts. Method: In vitro assays evaluated cell migration, colony formation, inhibition of the enzymatic activity in cell lysates, and combination therapy with gemcitabine. Result: There were mixed results; the inhibitors reduced the number of colonies but did not affect the total area. Cells migrated despite enzyme inhibition by Neq0709 and Neq0712. As expected, the compounds were non-cytotoxic; they improved the potency of gemcitabine in the combined therapy assay, especially for Neq0707. Conclusion: In summary, our findings revealed the complexity of dealing with the translation from biochemical to cell-based assays in the hit-to-lead step.

Contribution of cysteine and serine proteases to proteolytic digestion in an allergy-eliciting house dust mite

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.

Analysis of cathepsin B and cathepsin L treatment to clear toxic lysosomal protein aggregates in neuronal ceroid lipofuscinosis

Proteolysis mediated by lysosomal cathepsin proteases maintains a physiological flow in autophagy, phagocytosis and endocytosis. Neuronal Ceroid Lipofuscinosis (NCL) is a childhood neurodegenerative disorder characterized by disturbed autophagic flow and pathological accumulation of proteins. We demonstrated a therapeutic clearance of protein aggregates after dosing NCL10 mice with recombinant human pro-cathepsin-D. Prompted by these results and speculating that cathepsins may act in a redundant and in an hierarchical manner we envisaged that a treatment with human recombinant cysteine proteases pro-cathepsin-L (proCTSL) and pro-cathepsin-B (proCTSB) could similarly be used to induce protein degradation. Both enzymes were taken up by mannose 6-phosphate receptor- and LRP-receptor-mediated endocytosis and processed to the lysosomal mature cathepsins. In murine NCL10 astrocytes an abnormal increase in LAMP1 and saposin expression was revealed. Although proCTSB application did not improve this phenotype, proCTSL treatment led to reduced saposin-C levels in this model as well as in an acute brain slice model. Intracerebral dosing in a NCL10 mouse model revealed cellular and lysosomal uptake of both enzymes. Only proCTSL mildly reduced saposin-C levels and attenuated reactive astrogliosis. Application of both proteases did not improve weight loss and mortality of mutant mice. Our data reveal that although recombinant lysosomal proteases can be efficiently delivered to neuronal lysosomes cysteine proteases are less efficient in protein aggregates clearance as compared to the cathepsin-D treatment. Our data including in vitro degradation assays support the idea that bulk proteolysis requires a hierarchical process in which both aspartyl and cysteine hydrolases play a role.

Design, molecular docking, drug-likeness, and molecular dynamics studies of 1,2,4-trioxane derivatives as novel Plasmodium falciparum falcipain-2 (FP-2) inhibitors.

Despite significant progress made in drug discovery and development over the past few decades, malaria remains a life-threatening infectious disease across the globe. Because of the widespread emergence of drug-resistant strains of Plasmodium falciparum, the clinical utility of existing drug therapies including Artemisinin-based Combination Therapies (ACTs) in the treatment of malaria has been increasingly limited. It has become a serious health concern which, therefore, necessitates the development of novel drug molecules and/or alternative therapies to combat, particularly resistant P. falciparum. The objective of the present study was to develop 1,2,4-trioxane derivatives as novel antimalarial agents that would be effective against resistant P. falciparum. In our study, 15 new trioxane derivatives were designed by molecular modification of the 1,2,4-trioxane scaffold as possible antimalarial agents. Molecular modeling studies of trioxane derivatives were performed based on the CADD approach using Biovia Discovery Studio (DS) 2018 software. The protein-ligand docking study was performed against P. falciparum falcipain 2 (FP-2) using the simulation-based docking protocol LibDock by the flexible docking method. The assessment of drug-likeness, ADMET properties, and toxicity was also performed. Furthermore, the compounds CC3 and CC7, which showed the best binding affinity against the target P. falciparum FP-2, were investigated by molecular dynamics (MD) simulation studies followed by the calculation of MM-PBSA binding free energy of protein-ligand complexes using DS 2020. Results of the docking study showed that among the 15 compounds, three trioxane derivatives were found to possess promising binding affinity with LibDock scores ranging from 117.16 to 116.90. Drug-likeness, ADMET, and toxicity properties were found to be satisfactory for all the compounds. Among the 15 compounds, two compounds, namely CC3 and CC7, showed the highest binding affinity against FP-2 with LibDock score of 117.166 and 117.200, respectively. The Libdock score of the co-crystal inhibitor was 114.474. MD studies along with MM-PBSA calculations of binding energies further confirmed the antimalarial potential of the compounds CC3 and CC7, with the formation of well-defined and stable receptor-ligand interactions against the P. falciparum FP-2 enzyme. Additionally, the selectivity of trioxane hits identified as potential inhibitors of P. falciparum cysteine protease FP-2 was determined on human cysteine proteases such as cathepsins (Cat K and Cat L), which are host homologous. Finally, it was concluded that the newly designed 1,2,4-trioxane derivatives can be further studied for in vitro and in vivo antimalarial activities for their possible development as potent antimalarial agents effective against resistant P. falciparum.

Design, synthesis and stepwise optimization of nitrile-based inhibitors of cathepsins B and L

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e → 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure–activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Mapping the S1 and S1' subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 μM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.

ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease.

Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well characterized specificities to generate peptides amenable for identification by high-throughput tandem mass spectrometry. Trypsin, which cuts specifically after the basic residues lysine and arginine, is the predominant enzyme used for proteome digestion, although proteases with alternative specificities are required to detect sequences that are not accessible after tryptic digest. Here, we show that the human cysteine protease legumain exhibits a strict substrate specificity for cleavage after asparagine and aspartic acid residues during in-solution digestions of proteomes extracted from Escherichia coli, mouse embryonic fibroblast cell cultures, and Arabidopsis thaliana leaves. Generating peptides highly complementary in sequence, yet similar in their biophysical properties, legumain (as compared to trypsin or GluC) enabled complementary proteome and protein sequence coverage. Importantly, legumain further enabled the identification and enrichment of protein N-termini not accessible in GluC- or trypsin-digested samples. Legumain cannot cleave after glycosylated Asn residues, which enabled the robust identification and orthogonal validation of N-glycosylation sites based on alternating sequential sample treatments with legumain and PNGaseF and vice versa. Taken together, we demonstrate that legumain is a practical, efficient protease for extending the proteome and sequence coverage achieved with trypsin, with unique possibilities for the characterization of post-translational modification sites.

Structure-based Approaches Targeting Parasite Cysteine Proteases.

Cysteine proteases are essential hydrolytic enzymes present in the majority of organisms, including viruses and unicellular parasites. Despite the high sequence identity displayed among these proteins, specific structural features across different species grant distinct functions to these biomolecules, frequently related to pathological conditions. Consequently, their relevance as promising targets for potential specific inhibitors has been highlighted and occasionally validated in recent decades. In this review, we discuss the recent outcomes of structure-based campaigns aiming the discovery of new inhibitor prototypes against cruzain and falcipain, as alternative therapeutic tools for Chagas disease and malaria treatments, respectively. Computational and synthetic approaches have been combined on hit optimization strategies and are also discussed herein. These rationales are extended to additional tropical infectious and neglected pathologies, such as schistosomiasis, leishmaniasis and babesiosis, and also to Alzheimer's Disease, a widespread neurodegenerative disease poorly managed by currently available drugs and recently linked to particular physiopathological roles of human cysteine proteases.

Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Trypanosoma cruzi Cruzain.

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Deficiency of the human cysteine protease inhibitor cystatin M/E causes hypotrichosis and dry skin

PurposeWe aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. MethodsExome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. ResultsWe identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. ConclusionThe herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.