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.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

Therapeutic cysteine protease inhibitors: a patent review (2018-present).

INTRODUCTION: Cysteine proteases are involved in a broad range of biological functions, ranging from extracellular matrix turnover to immunity. Playing an important role in the onset and progression of several diseases, including cancer, immune-related and neurodegenerative disease, viral and parasitic infections, cysteine proteases represent an attractive drug target for the development of therapeutic tools. AREAS COVERED: Recent scientific and patent literature focusing on the design and study of cysteine protease inhibitors with potential therapeutic application has been reviewed. EXPERT OPINION: The discovery of a number of effective structurally diverse cysteine protease inhibitors opened up new challenges and opportunities for the development of therapeutic tools. Mechanistic studies and the availability of X-ray crystal structures of some proteases, alone and in complex with inhibitors, provide crucial information for the rational design and development of efficient and selective cysteine protease inhibitors as preclinical candidates for the treatment of different diseases.

Dipeptide-Derived Alkynes as Potent and Selective Irreversible Inhibitors of Cysteine Cathepsins.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogeneous products obtained in the Gilbert-Seyferth homologation for C≡C bond formation. Twenty-three dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S, and K. Numerous combinations of residues at positions P1 and P2 as well as terminal acyl groups allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of >3 orders of magnitude (3-10 133 M-1 s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

Falcipains: Biochemistry, target validation and structure-activity relationship studies of inhibitors as antimalarials.

Malaria is a tropical disease with significant morbidity and mortality burden caused by Plasmodium species in Africa, the Middle East, Asia, and South America. Pathogenic Plasmodium species have lately become increasingly resistant to approved chemotherapeutics and combination therapies. Therefore, there is an emergent need for identifying new druggable targets and novel chemical classes against the parasite. Falcipains, cysteine proteases required for heme metabolism in the erythrocytic stage, have emerged as promising drug targets against Plasmodium species that infect humans. This perspective discusses the biology, biochemistry, structural features, and genetics of falcipains. The efforts to identify selective or dual inhibitors and their structure-activity relationships are reviewed to give a perspective on the design of novel compounds targeting falcipains for antimalarial activity evaluating reasons for hits and misses for this important target.

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.

Experimental and Computational Study of Aryl-thiosemicarbazones Inhibiting Cruzain Reveals Reversible Inhibition and a Stepwise Mechanism.

Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi, is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1). Additionally, we studied a semicarbazone (compound 2), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The Ki was estimated to be 36.3 µM and Ki* to be 11.5 µM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S- on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1, involving the proton transfer to the ligand, followed by the Cys25-S- attack at C═S. The ΔG and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.

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.

Investigating the Lack of Translation from Cruzain Inhibition to Trypanosoma cruzi Activity with Machine Learning and Chemical Space Analyses.

Chagas disease is a neglected tropical disease caused by the protozoa Trypanosoma cruzi. Cruzain, its main cysteine protease, is commonly targeted in drug discovery efforts to find new treatments for this disease. Even though the essentiality of this enzyme for the parasite has been established, many cruzain inhibitors fail as trypanocidal agents. This lack of translation from biochemical to biological assays can involve several factors, including suboptimal physicochemical properties. In this work, we aim to rationalize this phenomenon through chemical space analyses of calculated molecular descriptors. These include statistical tests, visualization of projections, scaffold analysis, and creation of machine learning models coupled with interpretability methods. Our results demonstrate a significant difference between the chemical spaces of cruzain and T. cruzi inhibitors, with compounds with more hydrogen bond donors and rotatable bonds being more likely to be good cruzain inhibitors, but less likely to be active on T. cruzi. In addition, cruzain inhibitors seem to occupy specific regions of the chemical space that cannot be easily correlated with T. cruzi activity, which means that using predictive modeling to determine whether cruzain inhibitors will be trypanocidal is not a straightforward task. We believe that the conclusions from this work might be of interest for future projects that aim to develop novel trypanocidal compounds.

Study of the dynamic behavior of the cruzain enzyme in free and complexed forms with competitive and noncovalent benzimidazole inhibitors.

There are only two drugs for the treatment of Chagas disease, namely, nifurtimox and benznidazole, that can cause several adverse effects. Despite the effectiveness of these drugs in the disease's acute phase, they are not recognized as curative in the chronic phase, establishing the need for more effective treatment in all stages of the disease. Cruzain is an enzyme that plays a vital role in the life cycle of the etiologic agent, the protozoan Trypanosoma cruzi, being relevant as a therapeutic target in the planning of new drugs. Using molecular docking and dynamics simulations, we have investigated the structural and dynamic factors that can be involved in the enzyme inhibition process at the atomic-molecular level by benzimidazole compounds that are potent cruzain inhibitors with in vitro trypanocidal activity. The study suggests that these inhibitors bind cruzain through steric and hydrogen bonding interactions without altering its secondary structure content and protein compaction. Besides, we observed that these inhibitors decrease the correlation of movements between Cα-atoms of cruzain, increasing the number of atomic communities, mainly in the α-helix that presents the catalytic Cys25 residue. As expected, we also observed a correlation between the inhibitory activity of each inhibitor and their respective binding-free energies, reinforcing that the affinity of the complexes seems to be a relevant factor for enzymatic inhibition. Hence, the results presented in this work contribute to a better understanding of the cruzain enzyme inhibition mechanism through competitive and non-covalent inhibitors.Communicated by Ramaswamy H. Sarma.

Assessment of Reversibility for Covalent Cysteine Protease Inhibitors Using Quantum Mechanics/Molecular Mechanics Free Energy Surfaces.

We have used molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials to investigate the reaction mechanism for covalent inhibition of cathepsin K and assess the reversibility of inhibition. The computed free energy profiles suggest that a nucleophilic attack by the catalytic cysteine on the inhibitor warhead and proton transfer from the catalytic histidine occur in a concerted manner. The results indicate that the reaction is more strongly exergonic for the alkyne-based inhibitors, which bind irreversibly to cathepsin K, than for the nitrile-based inhibitor odanacatib, which binds reversibly. Gas-phase energies were also calculated for the addition of methanethiol to structural prototypes for a number of warheads of interest in cysteine protease inhibitor design in order to assess electrophilicity. The approaches presented in this study are particularly applicable to assessment of novel warheads, and computed transition state geometries can be incorporated into molecular models for covalent docking.

Lead optimization of cathepsin K inhibitors for the treatment of Osteoarthritis.

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10-SPR19) as potential anti-HAT agents. The new molecules show Ki values in the low-micro/sub-micromolar range against rhodesain, coupled with k2nd values between 1314 and 6950 M-1 min-1. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents.

Review: Unraveling the origin of the structural and functional diversity of plant cystatins.

The regulation of protease activity is a critical factor for the physiological balance during plant growth and development. Among the proteins involved in controlling protease activity are the cystatins, well-described inhibitors of cysteine proteases present in viruses, bacteria and most Eukaryotes. Plant cystatins, commonly called phytocystatins, display unique structural and functional diversity and are classified according to their molecular weight as type-I, -II, and -III. Their gene structure is highly conserved across Viridiplantae and provides insights into their evolutionary relationships. Many type-I phytocystatins with introns share sequence similarities with type-II phytocystatins. New data shows that they could have originated from recent losses of the carboxy-terminal extension present in type-II phytocystatins. Intronless type-I phytocystatins originated from a single event shared by flowering plants. Pieces of evidence show multiple events of gene duplications, intron losses, and gains throughout the expansion and diversity of the phytocystatin family. Gene duplication events in Gymnosperms and Eudicots resulted in inhibitors with amino acid substitutions that may modify their interaction with target proteases and other proteins. This review brings a phylogenomic analysis of plant cystatin evolution and contributes to a broader understanding of their origins. A complete functional genomic analysis among phytocystatins and their roles in plant development and responses to abiotic and biotic stresses remains a question to be fully solved.

New peptidomimetic rhodesain inhibitors with improved selectivity towards human cathepsins.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k2nd = 883 • 103 M-1 s-1) with single-digit nanomolar binding affinity (Ki = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis.

Development of novel dipeptide nitriles as inhibitors of rhodesain of Trypanosoma brucei rhodesiense.

In this paper, we developed a new series of dipeptide nitriles that were demonstrated to be reversible rhodesain inhibitors at nanomolar level, with EC50 values against cultured T. b. brucei in the micromolar range. We also proved that our dipeptide nitriles directly bind to the active site of rhodesain acting as competitive inhibitors. Within the most interesting compounds, the dipeptide nitrile 2b showed the highest binding affinity towards rhodesain (Ki = 16 nM) coupled with a good antiparasitic activity (EC50 = 14.1 µM). Moreover, for the dipeptide nitrile 3e, which showed a Ki = 122 nM towards the trypanosomal protease, we obtained the highest antiparasitic activity (EC50 = 8.8 µM). Thus, given the obtained results both compounds could certainly represent new lead compounds for the discovery of new drugs to treat Human African Trypanosomiasis.

Neoechinulin A as a Promising SARS-CoV-2 Mpro Inhibitor: In Vitro and In Silico Study Showing the Ability of Simulations in Discerning Active from Inactive Enzyme Inhibitors.

The COVID-19 pandemic and its continuing emerging variants emphasize the need to discover appropriate treatment, where vaccines alone have failed to show complete protection against the new variants of the virus. Therefore, treatment of the infected cases is critical. This paper discusses the bio-guided isolation of three indole diketopiperazine alkaloids, neoechinulin A (1), echinulin (2), and eurocristatine (3), from the Red Sea-derived Aspergillus fumigatus MR2012. Neoechinulin A (1) exhibited a potent inhibitory effect against SARS-CoV-2 Mpro with IC50 value of 0.47 µM, which is comparable to the reference standard GC376. Despite the structural similarity between the three compounds, only 1 showed a promising effect. The mechanism of inhibition is discussed in light of a series of extensive molecular docking, classical and steered molecular dynamics simulation experiments. This paper sheds light on indole diketopiperazine alkaloids as a potential structural motif against SARS-CoV-2 Mpro. Additionally, it highlights the potential of different molecular docking and molecular dynamics simulation approaches in the discrimination between active and inactive structurally related Mpro inhibitors.

Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues.

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Intramolecular Interactions Enhance the Potency of Gallinamide A Analogues against Trypanosoma cruzi.

Gallinamide A, a metabolite of the marine cyanobacterium Schizothrix sp., selectively inhibits cathepsin L-like cysteine proteases. We evaluated the potency of gallinamide A and 23 synthetic analogues against intracellular Trypanosoma cruzi amastigotes and the cysteine protease, cruzain. We determined the co-crystal structures of cruzain with gallinamide A and two synthetic analogues at ∼2 Å. SAR data revealed that the N-terminal end of gallinamide A is loosely bound and weakly contributes in drug-target interactions. At the C-terminus, the intramolecular π-π stacking interactions between the aromatic substituents at P1' and P1 restrict the bioactive conformation of the inhibitors, thus minimizing the entropic loss associated with target binding. Molecular dynamics simulations showed that in the absence of an aromatic group at P1, the substituent at P1' interacts with tryptophan-184. The P1-P1' interactions had no effect on anti-cruzain activity, whereas anti-T. cruzi potency increased by ∼fivefold, likely due to an increase in solubility/permeability of the analogues.

Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B.

CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.