Human Cathepsin Research Articles

Structure-based discovery of thiosemicarbazones as SARS-CoV-2 main protease inhibitors.

Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 μM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

In vitro validation of Cathepsin S docking models

Abstract In dendritic cells, the dominant lysosomal protease involved in Ii chain degradation and antigen digestion is Cathepsin S, a cysteine protease in the papain family. Extracellularly, Cathepsin S also contributes to pathological processes in cardiovascular disease, cancer, and autoimmune arthritis: unlike most lysosomal proteases, it retains its activity at serum pH. In computational molecular docking studies comparing peptides from the literature, we found that Cathepsin S shows a pH-dependent specificity switch, with predicted dissociation constants for specific peptide sequences differing at lysosomal and serum pH. The aim of this study is to further characterize this specificity switch and to compare docking results to kinetic parameters of Cathepsin S digestion in vitro. We identified peptide sequences of interest from docking, including sequences from the Ii chain and elastin, and digested these peptides (LifeTein) using human Cathepsin S (Calbiochem), collecting samples for LC-MS analysis over 16 hour reaction times. We report the kinetic parameters of Cathepsin S catalysis at both lysosomal and serum pH. Digestion results are compared to docking models and the models most predictive of catalytic activity are identified. Further investigation is needed to determine whether pH-dependent catalytic activity and selectivity of Cathepsin S will enable the design of protein-based therapies and inhibitors optimized for their targeted microenvironments. This work was partially supported by the NIDA/NIH award UG3DA048775, the USDA National Institute of Food and Agriculture, AFRI project #2021-0858, and the department of Biological Systems Engineering at Virginia Tech.

A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is theread out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

Homogeneous, Synthetic, Non-Saccharide Glycosaminoglycan Mimetics as Potent Inhibitors of Human Cathepsin G.

Cathepsin G (CatG) is a pro-inflammatory neutrophil serine protease that is important for host defense, and has been implicated in several inflammatory disorders. Hence, inhibition of CatG holds much therapeutic potential; however, only a few inhibitors have been identified to date, and none have reached clinical trials. Of these, heparin is a well-known inhibitor of CatG, but its heterogeneity and bleeding risk reduce its clinical potential. We reasoned that synthetic small mimetics of heparin, labeled as non-saccharide glycosaminoglycan mimetics (NSGMs), would exhibit potent CatG inhibition while being devoid of bleeding risks associated with heparin. Hence, we screened a focused library of 30 NSGMs for CatG inhibition using a chromogenic substrate hydrolysis assay and identified nano- to micro-molar inhibitors with varying levels of efficacy. Of these, a structurally-defined, octasulfated di-quercetin NSGM 25 inhibited CatG with a potency of ~50 nM. NSGM 25 binds to CatG in an allosteric site through an approximately equal contribution of ionic and nonionic forces. Octasulfated 25 exhibits no impact on human plasma clotting, suggesting minimal bleeding risk. Considering that octasulfated 25 also potently inhibits two other pro-inflammatory proteases, human neutrophil elastase and human plasmin, the current results imply the possibility of a multi-pronged anti-inflammatory approach in which these proteases are likely to simultaneously likely combat important conditions, e.g., rheumatoid arthritis, emphysema, or cystic fibrosis, with minimal bleeding risk.

Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.

Impact of the Recognition Part of Dipeptidyl Nitroalkene Compounds on the Inhibition Mechanism of Cysteine Proteases Cruzain and Cathepsin L.

Cysteine proteases (CPs) are an important class of enzymes, many of which are responsible for several human diseases. For instance, cruzain of protozoan parasite Trypanosoma cruzi is responsible for the Chagas disease, while the role of human cathepsin L is associated with some cancers or is a potential target for the treatment of COVID-19. However, despite paramount work carried out during the past years, the compounds that have been proposed so far show limited inhibitory action against these enzymes. We present a study of proposed covalent inhibitors of these two CPs, cruzain and cathepsin L, based on the design, synthesis, kinetic measurements, and QM/MM computational simulations on dipeptidyl nitroalkene compounds. The experimentally determined inhibition data, together with the analysis and the predicted inhibition constants derived from the free energy landscape of the full inhibition process, allowed describing the impact of the recognition part of these compounds and, in particular, the modifications on the P2 site. The designed compounds and, in particular, the one with a bulky group (Trp) at the P2 site show promising in vitro inhibition activities against cruzain and cathepsin L for use as a starting lead compound in the development of drugs with medical applications for the treatment of human diseases and future designs.

Abstract LB221: Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses

Abstract The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Emma Renard, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB221.

Abstract LB219: Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation

Abstract The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using a Topoisomerase 1 (Topo1) inhibitor as payload with highly favorable biophysical properties and superior anti-tumor efficacy compared to Trastuzumab deruxtecan in head-to-head in vitro and in vivo studies. Based on trastuzumab as the targeting antibody and a Topoisimerase 1 inhibitor as payload, we generated highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of 2. In in-vitro assays on target positive cell-lines, the Araris Topo 1 ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved Trastuzumab deruxtecan which has a DAR of 8. Moreover, the ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while Trastuzumab deruxtecan showed significant payload loss during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for payload release was highly efficient in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile similar to the unmodified trastuzumab parent antibody. In efficacy studies using an established NCI-N87 colon cancer model (therapeutic setting), a single injection of the Araris Topo 1 ADC at DAR2 at a dose of 52ug/kg (adjusted payload dose) induced superior anti-tumor activity compared to Trastuzumab deruxtecan at DAR of 8, injected at the same payload dose. Complete tumor regression of all tumors (7/7) was obtained at 104ug/kg payload dose and lasted throughout the whole study duration (total 80 days) and was very well tolerated. The data show that Araris Topo 1 ADCs assembled using novel peptide linkers, even at a DAR of as low as 2 have a very efficient anti-tumor activity suggesting optimal drug exposure, targeting and release of the payload. In summary, we show that the Araris Topo1 linker-payloads result in highly potent ADCs with very favorable biophysical properties and extremely efficient payload release as well as an antibody-like exposure profile making them ideal linker-payloads for solid tumor targeting. We anticipate the low-drug load to be favorable in avoiding excessive toxicities in non-targeted tissues. Finally, the Araris bioconjugation technology allows for the generation of tailor-made ADC candidates with improved therapeutic indices. Citation Format: Isabella Attinger-Toller, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB219.

Structure-Based Optimization of Protease-Inhibitor Interactions to Enhance Specificity of Human Stefin-A against Falcipain-2 from the Plasmodium falciparum 3D7 Strain.

The emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies has raised global concerns and emphasized the identification of new drug targets for malaria. Cysteine protease falcipain-2 (FP2), involved in host hemoglobin degradation and instrumental in parasite survival, has long been proposed as a promising malarial drug target. However, designing active-site-targeted small-molecule inhibitors of FP2 becomes challenging due to their off-target specificity toward highly homologous human cysteine cathepsins. The use of proteinaceous inhibitors, which have nonconserved exosite interactions and larger interface area, can effectively circumvent this problem. In this study, we report for the first time that human stefin-A (STFA) efficiently inhibits FP2 with Ki values in the nanomolar range. The FP2-STFA complex crystal structure, determined in this study, and sequence analyses identify a unique nonconserved exosite interaction, compared to human cathepsins. Designing a mutation Lys68 > Arg in STFA amplifies its selectivity garnering a 3.3-fold lower Ki value against FP2, and the crystal structure of the FP2-STFAK68R complex shows stronger electrostatic interaction between side-chains of Arg68 (STFAK68R) and Asp109 (FP2). Comparative structural analyses and molecular dynamics (MD) simulation studies of the complexes further confirm higher buried surface areas, better interaction energies for FP2-STFAK68R, and consistency of the newly developed electrostatic interaction (STFA-R68-FP2-D109) in the MD trajectory. The STFA-K68R mutant also shows higher Ki values against human cathepsin-L and stefin, a step toward eliminating off-target specificity. Hence, this work underlines the design of host-based proteinaceous inhibitors against FP2, with further optimization to render them more potent and selective.

Rhenium(V) Complexes as Cysteine-Targeting Coordinate Covalent Warheads.

Interest in covalent enzyme inhibitors as therapeutic agents has seen a recent resurgence. Covalent enzyme inhibitors typically possess an organic functional group that reacts with a key feature of the target enzyme, often a nucleophilic cysteine residue. Herein, the application of small, modular ReV complexes as inorganic cysteine-targeting warheads is described. These metal complexes were found to react with cysteine residues rapidly and selectively. To demonstrate the utility of these ReV complexes, their reactivity with SARS-CoV-2-associated cysteine proteases is presented, including the SARS-CoV-2 main protease and papain-like protease and human enzymes cathepsin B and L. As all of these proteins are cysteine proteases, these enzymes were found to be inhibited by the ReV complexes through the formation of adducts. These findings suggest that these ReV complexes could be used as a new class of warheads for targeting surface accessible cysteine residues in disease-relevant target proteins.

In silico Analysis and Characterization of a Putative Aspartic Proteinase Inhibitor, IA3 from Lachancea kluyveri

Aspartic proteinases play important role in various physiological and biological processes. Understanding catalytic mechanisms of these enzymes could lead to crucial medical and biological applications. Two types of aspartic proteinase inhibitors have been identified i.e. small molecule inhibitor and naturally occurring peptides. Most of aspartic proteinases are highly susceptible to inhibition by a series of small, non-proteinaceous natural products; pepstatin. However, only limited number of naturally-occurring polypeptide inhibitors of aspartic proteinases has so far been discovered. One among these inhibitors is Saccharomyces cerevisiae IA3. The S. cerevisiae Proteinase A (ScPrA) is solely and potently inhibited by this small polypeptide at sub-nanomolar level. It was proven that, not only IA3 has no detectable effect against a wide range of aspartic proteinases, but it was also shown to be cleaved as a substrate of these non-target enzymes. Bioinformatics analysis of the IA3 structure has been undertaken in this study. Database searching for sequence homology from available fungal genome data bank using the Basic Local Alignment Search Tool (BLAST) revealed that 4 structurally related, IA3-like proteins have successfully been identified. The amino acid sequence of IA3-like proteins from Lachancea kluyveri share highest degree of similarity toward wild-type IA3. The Lk-IA3-like gene was synthesized using a PCR-based gene synthesis method. Protein expression in E. coli, protein purification and characterization of Lk-IA3-like by enzyme kinetic assays were performed. The results indicated that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM and possesses no inhibitory effect toward AfPrA (Aspergillus fumigatus proteinase A) or HuCatD (Human cathepsin D). HIGHLIGHTS Aspartic proteinases enzyme family is crucially involved in various physiological and biological processes including in the pathogenesis of numerous diseases which make them a possible target for drug design Inhibitors of aspartic proteinases are not only applied for human disease treatment but also extended to plant and other economically important animals Aspartic proteinase from cerevisiae (Proteinase A) is solely and potently inhibited by a small peptideinhibitor, IA3 Bioinformatics analysis of the naturally occurring aspartic proteinases inhibitor, IA3 structure has been undertaken in this research and putative IA3-like proteins have successfully been identified IA3-like proteins from Lachancea kluyveri were produced in coli and enzyme inhibition assays revealed that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM GRAPHICAL ABSTRACT

Cathepsin K: both a likely biomarker and a new therapeutic target in lymphangioleiomyomatosis?

Lymphangioleiomyomatosis (LAM) is a rare disease which is characterized by cystic lung destruction and lymphangiomas, and is associated with a high risk of osteoporosis-related bone fractures. Its diagnosis is based on pulmonary anatomopathological criteria combined with chest computed tomography. VEGF-D is the only serum diagnostic biomarker used in clinic, while inhibition of the mTOR pathway by rapamycin is currently the only reference therapy for LAM. Human cathepsin K (CatK), a potent collagenase predominantly found in osteoclasts, is considered as a valuable target for anti-osteoporosis and bone cancer therapy. Recently, CatK, which is overexpressed in lung cysts, was proposed as a putative LAM biomarker. Moreover, CatK may take part in the LAM pathophysiology by participating in pulmonary cystic destruction and bone degradation. Accordingly, targeting of collagenolytic activity of CatK by exosite-binding inhibitors in combination with mTOR inhibition could represent an innovative therapeutic option in reducing lung destruction in LAM.

Computational insights for predicting the binding and selectivity of peptidomimetic plasmepsin IV inhibitors against cathepsin D.

Plasmepsins (Plms) are aspartic proteases involved in the degradation of human hemoglobin by P. falciparum and are essential for the survival and growth of the parasite. Therefore, Plm enzymes are reported as an important antimalarial drug target. Herein, we have applied molecular docking, molecular dynamics (MD) simulations, and binding free energy with the Linear Interaction Energy (LIE) approach to investigate the binding of peptidomimetic PlmIV inhibitors with a particular focus on understanding their selectivity against the human Asp protease cathepsin D (CatD). The residual decomposition analysis results suggest that amino acid differences in the subsite S3 of PlmIV and CatD are responsible for the higher selectivity of the 5a inhibitor. These findings yield excellent agreement with experimental binding data and provide new details regarding van der Waals and electrostatic interactions of subsite residues as well as structural properties of the PlmIV and CatD systems.

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Bridging the Gap in Malaria Parasite Resistance, Current Interventions, and the Way Forward from in Silico Perspective: A Review.

The past decade has seen most antimalarial drugs lose their clinical potency stemming from parasite resistance. Despite immense efforts by researchers to mitigate this global scourge, a breakthrough is yet to be achieved, as most current malaria chemotherapies suffer the same fate. Though the etiology of parasite resistance is not well understood, the parasite's complex life has been implicated. A drug-combination therapy with artemisinin as the central drug, artemisinin-based combination therapy (ACT), is currently the preferred malaria chemotherapy in most endemic zones. The emerging concern of parasite resistance to artemisinin, however, has compromised this treatment paradigm. Membrane-bound Ca2+-transporting ATPase and endocytosis pathway protein, Kelch13, among others, are identified as drivers in plasmodium parasite resistance to artemisinin. To mitigate parasite resistance to current chemotherapy, computer-aided drug design (CADD) techniques have been employed in the discovery of novel drug targets and the development of small molecule inhibitors to provide an intriguing alternative for malaria treatment. The evolution of plasmepsins, a class of aspartyl acid proteases, has gained tremendous attention in drug discovery, especially the non-food vacuole. They are expressed at multi-stage of the parasite's life cycle and involve in hepatocytes' egress, invasion, and dissemination of the parasite within the human host, further highlighting their essentiality. In silico exploration of non-food vacuole plasmepsin, PMIX and PMX unearthed the dual enzymatic inhibitory mechanism of the WM382 and 49c, novel plasmepsin inhibitors presently spearheading the search for potent antimalarial. These inhibitors impose structural compactness on the protease, distorting the characteristic twist motion. Pharmacophore modeling and structure activity of these compounds led to the generation of hits with better affinity and inhibitory prowess towards PMIX and PMX. Despite these headways, the major obstacle in targeting PM is the structural homogeneity among its members and to human Cathepsin D. The incorporation of CADD techniques described in the study at early stages of drug discovery could help in selective inhibition to augment malaria chemotherapy.

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

Potent and biostable inhibitors of the main protease of SARS-CoV-2

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection invitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O-glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

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.