Structural Elucidation and Antiviral Activity of Covalent Cathepsin L Inhibitors.

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.

Establishment, optimization and validation of a fluorescence polarization-based high-throughput screening assay targeting cathepsin L inhibitors.

Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.

SiteMine: Large-scale binding site similarity searching in protein structure databases.

Drug discovery and design challenges, such as drug repurposing, analyzing protein-ligand and protein-protein complexes, ligand promiscuity studies, or function prediction, can be addressed by protein binding site similarity analysis. Although numerous tools exist, they all have individual strengths and drawbacks with regard to run time, provision of structure superpositions, and applicability to diverse application domains. Here, we introduce SiteMine, an all-in-one database-driven, alignment-providing binding site similarity search tool to tackle the most pressing challenges of binding site comparison. The performance of SiteMine is evaluated on the ProSPECCTs benchmark, showing a promising performance on most of the data sets. The method performs convincingly regarding all quality criteria for reliable binding site comparison, offering a novel state-of-the-art approach for structure-based molecular design based on binding site comparisons. In a SiteMine showcase, we discuss the high structural similarity between cathepsin L and calpain 1 binding sites and give an outlook on the impact of this finding on structure-based drug design. SiteMine is available at https://uhh.de/naomi.

Nanoengineered Red Blood Cells Loaded with TMPRSS2 and Cathepsin L Inhibitors Block SARS-CoV-2 Pseudovirus Entry into Lung ACE2+ Cells.

The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.

Olgotrelvir, a dual inhibitor of SARS-CoV-2 Mpro and cathepsin L, as a standalone antiviral oral intervention candidate for COVID-19.

BACKGROUND: Oral antiviral drugs with improved antiviral potency and safety are needed to address current challenges in clinical practice for treatment of COVID-19, including the risks of rebound, drug-drug interactions, and emerging resistance. METHODS: Olgotrelvir (STI-1558) is designed as a next-generation antiviral targeting the SARS-CoV-2 main protease (Mpro), an essential enzyme for SARS-CoV-2 replication, and human cathepsin L (CTSL), a key enzyme for SARS-CoV-2 entry into host cells. FINDINGS: Olgotrelvir is a highly bioavailable oral prodrug that is converted in plasma to its active form, AC1115. The dual mechanism of action of olgotrelvir and AC1115 was confirmed by enzyme activity inhibition assays and co-crystal structures of AC1115 with SARS-CoV-2 Mpro and human CTSL. AC1115 displayed antiviral activity by inhibiting replication of all tested SARS-CoV-2 variants in cell culture systems. Olgotrelvir also inhibited viral entry into cells using SARS-CoV-2 Spike-mediated pseudotypes by inhibition of host CTSL. In the K18-hACE2 transgenic mouse model of SARS-CoV-2-mediated disease, olgotrelvir significantly reduced the virus load in the lungs, prevented body weight loss, and reduced cytokine release and lung pathologies. Olgotrelvir demonstrated potent activity against the nirmatrelvir-resistant Mpro E166 mutants. Olgotrelvir showed enhanced oral bioavailability in animal models and in humans with significant plasma exposure without ritonavir. In phase I studies (ClinicalTrials.gov: NCT05364840 and NCT05523739), olgotrelvir demonstrated a favorable safety profile and antiviral activity. CONCLUSIONS: Olgotrelvir is an oral inhibitor targeting Mpro and CTSL with high antiviral activity and plasma exposure and is a standalone treatment candidate for COVID-19. FUNDING: Funded by Sorrento Therapeutics.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation.

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

ASPER-29 suppresses the metastasis of pancreatic cancer cells by dual inhibition of cathepsin-L and cathepsin-S.

Pancreatic cancer will be the second leading cause of cancer-related mortality worldwide due to its high rate of metastasis. Cathepsins (CATs) are effectors of invasive growth in various cancers. Currently, targeting CATs represents an attractive strategy for the treatment of highly metastatic cancers with high CATs activity, such as pancreatic cancer. To develop a stronger antimetastatic agent, ASPER-29, a novel inhibitor of CATs designed by using the asperphenamate derivative BBP as a lead compound, was synthesized, and its therapeutic potential in pancreatic cancer metastasis was investigated in this study. Molecular docking and enzyme inhibition assays proved that ASPER-29 can inhibit the activity of CAT-L and CAT-S by binding with these enzymes in classical action modes. Furthermore, ASPER-29 significantly inhibited the activity of CAT-L and CAT-S but had no effect on their expression in PANC-1 and BxPC-3 cells. The in vitro antimetastatic activities of ASPER-29 were examined by wound healing and Transwell chamber assays. We found that ASPER-29 inhibited the migration and invasion of PANC-1 and BxPC-3 cells in a concentration-dependent manner. Moreover, the in vivo antimetastatic effects of ASPER-29 were confirmed in a mouse xenotransplantation model. H&E staining and immunohistochemistry assays of Ki67 and CEACAM6 proved that ASPER-29 treatment significantly blocked the metastasis of pancreatic cancer cells to lung and liver tissues. Additionally, the activity of both CAT-L and CAT-S was markedly inhibited in the lung and liver tissues of ASPER-29-administered mice compared with the mice in the model group, suggesting that the metastasis-blocking effect of ASPER-29 should be mediated via inhibition of the activity of CAT-L and CAT-S in pancreatic cancer cells. Together, our results demonstrated that ASPER-29, as a novel inhibitor of CAT-L and CAT-S, possessed the evident ability to block the metastasis of pancreatic cancer cells.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L.

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

MPI8 is Potent against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L.

A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro ) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10-3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

XPA is susceptible to proteolytic cleavage by cathepsin L during lysis of quiescent cells.

The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3-4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.

Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity.

The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.

An efficient and concise synthesis of a selective small molecule non-peptide inhibitor of cathepsin L: KGP94.

KGP94 is a potent, selective, and competitive inhibitor of the lysosomal endopeptidase enzyme (Cathepsin L) currently in preclinical trials for the treatment of metastatic cancer, which is a leading cause of cancer-associated death. Herein, we report two new synthetic routes for synthesizing the target compound through four consecutive steps, using a Weinreb amide approach starting from a common 3-bromobenzoyl chloride. A key step in the approach is a coupling reaction of a readily available Grignard reagent with amide 4 to produce 6, a previously unreported coupling pattern. These new strategies offer an efficient and alternative approach to synthesis of target compound with an excellent overall yield.

Discovery of a novel and selective cathepsin L inhibitor with anti-metastatic ability in vitro and in vivo against breast cancer cells.

Asperphenamate is a natural product that has attracted considerable interest from researchers worldwide. In the last decade, aiming to increase the biological activity and improve druggability, modifications of the A-ring moiety in asperphenamate have been performed. Our laboratory has also recently reported functional derivatizations of the A ring and studied its effect on the inhibition of cysteine cathepsin L. However, the functional significance of the B-ring fragment toward cathepsin L has not been evaluated thus far. In this paper, forty-four derivatives of the B-ring substituted with different N-phenylsulfonyl groups were designed and synthesized. Among them, the paratrifluromethyl analog B-2a and the 2, 4-difluoro-5-chloro derivative B-11b showed more potent inhibitory activity against cathepsin L than the control compound, ABR, which displayed the strongest inhibitory effect on cathepsin L and S among all reported asperphenamate derivatives. In particular, compound B-2a showed more pronounced selectivity against cathepsin L than the other derivatives. Molecular docking revealed that the N-phenylsulfonylamide moiety was vital for the interactions between B-2a and cathepsin L. Moreover, B-2a displayed no toxicity against normal cells. Therefore, compound B-2a was selected for further studies. Wound-healing assays, Transwell chamber assays and breast cancer lung metastasis mouse models demonstrated that B-2a exhibited antimetastatic ability in vitro and in vivo.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Cancer-associated mutations reveal a novel role for EpCAM as an inhibitor of cathepsin-L and tumor cell invasion.

BACKGROUND: EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors. METHODS: The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants. RESULTS: We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion. CONCLUSIONS: These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Molecular modeling for potential cathepsin L inhibitor identification as new anti-photoaging agents from tropical medicinal plants.

Ultraviolet exposure can cause photoaging toward the human skin which is begun by the inflammation on the exposure area, also resulting in activation of a degradative enzyme cathepsin L. This enzyme is one of the interesting novel therapeutic targets for antiaging agents. Three plants, named Kleinhovia hospita, Aleurites moluccana, and Centella asiatica, are well-known in the tropical region as anti-inflammatory herbs. The aims of this study were to predict the antiaging activity of the 31 compounds from these plants via inhibition of cathepsin L. All compounds were minimized their energies and then used in molecular docking. After that, molecular dynamics (MD) simulation was employed for the 5 candidate ligands and the positive control; schinol. Interaction analysis results of the pre-MD and post-MD simulation structures were obtained. Furthermore, a toxicity test was performed using ADMET Predictor 7.1. Based on the molecular docking and the MD simulation results, kleinhospitine A, ß-amyrin, and castiliferol exhibited lower binding free energy than schinol (-27.0925, -28.6813, -26.0037 kcal/mol) and also had interactions with the S´ region binding site. The toxicity test indicated that ß-amyrin is the most potential candidate since it exhibited the lowest binding energy and the high safety level.

ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model.

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development.

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.