Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

Nitriles: an attractive approach to the development of covalent inhibitors.

Nitriles have broad applications in medicinal chemistry, with more than 60 small molecule drugs on the market containing the cyano functional group. In addition to the well-known noncovalent interactions that nitriles can perform with macromolecular targets, they are also known to improve drug candidates' pharmacokinetic profiles. Moreover, the cyano group can be used as an electrophilic warhead to covalently bind an inhibitor to a target of interest, forming a covalent adduct, a strategy that can present benefits over noncovalent inhibitors. This approach has gained much notoriety in recent years, mainly with diabetes and COVID-19-approved drugs. Nevertheless, the application of nitriles in covalent ligands is not restricted to it being the reactive center, as it can also be employed to convert irreversible inhibitors into reversible ones, a promising strategy for kinase inhibition and protein degradation. In this review, we introduce and discuss the roles of the cyano group in covalent inhibitors, how to tune its reactivity and the possibility of achieving selectivity only by replacing the warhead. Finally, we provide an overview of nitrile-based covalent compounds in approved drugs and inhibitors recently described in the literature.

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.

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.

A patent review on cathepsin K inhibitors to treat osteoporosis (2011 - 2021).

INTRODUCTION: Cathepsin K (CatK) is a lysosomal cysteine protease and the predominant cathepsin expressed in osteoclasts, where it degrades the bone matrix. Hence, CatK is an attractive therapeutic target related to diseases characterized by bone resorption, like osteoporosis. AREAS COVERED: This review summarizes the patent literature from 2011 to 2021 on CatK inhibitors and their potential use as new treatments for osteoporosis. The inhibitors were classified by their warheads, with the most explored nitrile-based inhibitors. Promising in vivo results have also been disclosed. EXPERT OPINION: As one of the most potent lysosomal proteins whose primary function is to mediate bone resorption, cathepsin K remains an excellent target for therapeutic intervention. Nevertheless, there is no record of any approved drug that targets CatK. The most notable cases of drug candidates targeting CatK were balicatib and odanacatib, which reached Phase II and III clinical trials, respectively, but did not enter the market. Further developments include exploring new chemical entities beyond the nitrile-based chemical space, with improved ADME and safety profiles. In addition, CatK's role in cancer immunoexpression and its involvement in the pathophysiology of osteo- and rheumatoid arthritis have raised the race to develop activity-based probes with excellent potency and selectivity.

Predicting the Relative Binding Affinity for Reversible Covalent Inhibitors by Free Energy Perturbation Calculations.

Covalent inhibitors are assuming central importance in drug discovery projects, especially in this pandemic scenario. Many research groups have focused their attention on inhibiting viral proteases or human proteases such as cathepsin L (hCatL). The inhibition of these critical enzymes may impair viral replication. However, molecular modeling of covalent ligands is challenging since covalent and noncovalent ligand-bound states must be considered in the binding process. In this work, we evaluated the suitability of free energy perturbation (FEP) calculations as a tool for predicting the binding affinity of reversible covalent inhibitors of hCatL. Our strategy relies on the relative free energy calculated for both covalent and noncovalent complexes and the free energy changes have been compared with experimental data for eight nitrile-based inhibitors, including three new inhibitors of hCatL. Our results demonstrate that the covalent complex can be employed to properly rank the inhibitors. Nevertheless, a comparison of the free energy changes in both noncovalent and covalent states is valuable to interpret the effect triggered by the formation of the covalent bond on the interactions played by functional groups distant from the warhead. Overall, FEP can be employed as a powerful predictor tool in developing and understanding the activity of reversible covalent inhibitors.

Nitrile-based peptoids as cysteine protease inhibitors.

Peptidomimetics of the class of dipeptidyl nitrile analog peptoids were synthesized as inhibitors of mammalian cysteine proteases of the papain superfamily. The dipeptidyl nitrile side chains were attached to the peptide backbone's nitrogen atom, not to the α-carbons. Synthesized nitrile-based peptoid analogs that lack the hydrogen amide at P2-P3 are responsible for many of the secondary structure elements in peptides and proteins, making them resistant to proteolysis. The designed peptoids would lose a hydrogen bond with cruzain Asp161 decreasing the affinity toward the enzyme. A structure-activity relationship and matched molecular pair-based analysis between the dipeptidyl nitrile Neq0409 and its peptoid 4a yielded the following cruzain affinities: pKiNeq0409 = 6.5 and pKi4a = 5.2. respectively. A retrosynthetic matched molecular pair cliff (RMMP-cliff) analysis with a ΔpKiNeq0409-4a of 1.3 log is found for this transformation. These novel peptoids were then optimized, leading to compound 4i, with high cruzain inhibition (pKi = 6.8). Cross-class cathepsin activity was observed for some of these novel compounds against cathepsins K, L and S, while other compounds presented a selective inhibition of cathepsin K (4b, 4c, 4k) over ten times higher than the other enzymes. The putative mode of binding was determined by using covalent docking, which also aided to describe the structure-activity relationship (SAR). Interestingly, none of the peptoids inhibited CatB to any appreciable extent. These results provide guidance to identify novel bioactive nitrile-based peptoids.

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

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

Dipeptidyl nitrile derivatives suppress the Trypanosoma cruzi in vitro infection.

Chagas disease affects several countries around the world with health and sanitation problems. Cysteine proteases are essential for the virulence and replication of the Trypanosoma cruzi, being modulated by dipeptidyl nitriles and derivatives. Here, four dipeptidyl nitrile derivatives were assayed in three T. cruzi morphologies and two strains (Tulahuen and Y) using a set of assays: (i) analysis of the inhibitory activity against cysteine proteases; (ii) determination of the cytotoxic activity and selectivity index; (iii) verification of the inhibition of the trypomastigote invasion in the host cell. These compounds could inhibit the activity of cysteine proteases using the selective substrate Z-FR-MCA for the trypomastigote lysate and extracellular amastigotes. Interestingly, these compounds did not present relevant enzymatic inhibition for the epimastigote lysate. Most of the substances were also cytotoxic and selective against the trypomastigotes and intracellular amastigotes. The best compound of the series (Neq0662) could reduce the enzymatic activity of the cysteine proteases for the trypomastigotes and amastigotes. It was equipotent to the benznidazole drug in the cytotoxic studies using these two parasite forms. Neq0662 was also selective for the parasite, and it inhibited the invasion of the mammalian host cell in all conditions tested at 10 µM. The stereochemistry of the trifluoromethyl group was an important factor for the bioactivity when the two diastereomers (Neq0662 and Neq0663) were compared. All-in-all, these results indicate that these compounds could move further in the drug development stage because of its promising bioactive profile.

Molecular design aided by random forests and synthesis of potent trypanocidal agents as cruzain inhibitors for Chagas disease treatment.

Cruzain is an established target for the identification of novel trypanocidal agents, but how good are in vitro/in vivo correlations? This work describes the development of a random forests model for the prediction of the bioavailability of cruzain inhibitors that are Trypanosoma cruzi killers. Some common properties that characterize drug-likeness are poorly represented in many established cruzain inhibitors. This correlates with the evidence that many high-affinity cruzain inhibitors are not trypanocidal agents against T. cruzi. On the other hand, T. cruzi killers that present typical drug-like characteristics are likely to show better trypanocidal action than those without such features. The random forests model was not outperformed by other machine learning methods (such as artificial neural networks and support vector machines), and it was validated with the synthesis of two new trypanocidal agents. Specifically, we report a new lead compound, Neq0565, which was tested on T. cruzi Tulahuen (ß-galactosidase) with a pEC50 of 4.9. It is inactive in the host cell line showing a selectivity index (SI = EC50cyto /EC50T. cruzi ) higher than 50.

Crystal structure of Leishmania mexicana cysteine protease B in complex with a high-affinity azadipeptide nitrile inhibitor.

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.

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

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

Optimization strategy of single-digit nanomolar cross-class inhibitors of mammalian and protozoa cysteine proteases.

Cysteine proteases (CPs) are involved in a myriad of actions that include not only protein degradation, but also play an essential biological role in infectious and systemic diseases such as cancer. CPs also act as biomarkers and can be reached by active-based probes for diagnostic and mechanistic purposes that are critical in health and disease. In this paper, we present the modulation of a CP panel of parasites and mammals (Trypanosoma cruzi cruzain, LmCPB, CatK, CatL and CatS), whose inhibition by nitrile peptidomimetics allowed the identification of specificity and selectivity for a given CP. The activity cliffs identified at the CP inhibition level are useful for retrieving trends through multiple structure-activity relationships. For two of the cruzain inhibitors (10g and 4e), both enthalpy and entropy are favourable to Gibbs binding energy, thus overcoming enthalpy-entropy compensation (EEC). Group contribution of individual molecular modification through changes in enthalpy and entropy results in a separate partition on the relative differences of Gibbs binding energy (ΔΔG). Overall, this study highlights the role of CPs in polypharmacology and multi-target screening, which represents an imperative trend in the actual drug discovery effort.

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

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

Assessment of the Cruzain Cysteine Protease Reversible and Irreversible Covalent Inhibition Mechanism.

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol-1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.

Discovery of small molecule inhibitors of Leishmania braziliensis Hsp90 chaperone.

Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.

Evaluating QM/MM Free Energy Surfaces for Ranking Cysteine Protease Covalent Inhibitors.

One tactic for cysteine protease inhibition is to form a covalent bond between an electrophilic atom of the inhibitor and the thiol of the catalytic cysteine. In this study, we evaluate the reaction free energy obtained from a hybrid quantum mechanical/molecular mechanical (QM/MM) free energy profile as a predictor of affinity for reversible, covalent inhibitors of rhodesain. We demonstrate that the reaction free energy calculated with the PM6/MM potential is in agreement with the experimental data and suggest that the free energy profile for covalent bond formation in a protein environment may be a useful tool for the inhibitor design.

On the intrinsic reactivity of highly potent trypanocidal cruzain inhibitors.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we evaluated how different warheads for a set of structurally similar related compounds could inhibit the activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then, we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain inhibitor Neq0656 as a better trypanocidal agent (ECY.strain 50 = 0.1 µM; SI = 58.4) than the current drug benznidazole (ECY.strain 50 = 5.1 µM; SI > 19.6). We also measured the half-life time by HPLC analysis of three lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile), where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process.

Ligand-induced conformational selection predicts the selectivity of cysteine protease inhibitors.

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.

Predicting the affinity of halogenated reversible covalent inhibitors through relative binding free energy.

Nitrile reversible covalent inhibitors of human cathepsin L (hCatL) bind covalently to the side chain of the catalytic Cys25 residue in the S1 pocket to form thioimidates. Predicting the binding of reversible covalent inhibitors is essential for their practical application in drug design. In this report, five nitrile-based inhibitors coded Neq0570, Neq0710, Neq0802, Neq0803 and Neq0804 had their hCatL inhibition constants, Ki, determined. These analogs of the prototypical Neq0570 are halogenated reversible covalent inhibitors of hCatL, which bear a halogen atom in the meta position of the P3 benzyl ring that can form a halogen bond with the Gly61 of the hCatL. To describe halogen bonding interaction in an inhibitor-hCatL complex, we applied an extra point (EP) of charge to represent the anisotropic distribution of charge on the iodine, bromine and chlorine atoms. Besides, we have used alchemical free energy calculations for evaluating the overall relative binding free energies of these inhibitors using a two-state binding model: noncovalent and covalent bond states. Our results show that free energy perturbation (FEP) can predict the hCatL binding affinities of halogenated reversible covalent inhibitors in close agreement with experiments.