Optimization of Triarylpyridinone Inhibitors of the Main Protease of SARS-CoV-2 to Low-Nanomolar Antiviral Potency
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99 GENERAL AND MISCELLANEOUS↗
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Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents
SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P 2 position of a peptidomimetic inhibitor. At the P 1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P 1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from K i = 0.6–18 nM (cathepsin L) and K i = 2.6–124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC 50 = 0.47–15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys 145 , and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P 3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.
60 APPLIED LIFE SCIENCES↗
Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro
SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-11 polyprotein, whose mature products include cofactors of the viral replicase, and identified the order of cleavages. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and x-ray scattering yielded a nsp7-11 structural ensemble, demonstrating shared secondary structural elements with individual nsps. The pattern of cross-links and HDX footprint of the C145A Mpro and nsp7-11 complex demonstrate preferential binding of the enzyme active site to the polyprotein junction sites and additional transient contacts to help orient the enzyme on its substrate for cleavage. Last, proteolysis assays were used to characterize the effect of inhibitors/binders on Mpro processing/inhibition using the nsp7-11 polyprotein as substrate.
60 APPLIED LIFE SCIENCES↗
Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2
Targeting the main protease of SARS-CoV-2 Inside host cells, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated into two polyproteins that are cleaved to give the individual viral proteins. The main viral protease, known as Mpro or 3CLpro, plays a key role in these cleavages, making it an important drug target. Drayman et al . identified eight drugs that target 3CLpro from a library of 1900 clinically safe drugs. Because of the challenge of working with SARS-CoV-2, they started by screening for drugs that inhibit the replication of a human coronavirus that causes the common cold. They then evaluated the top hits for inhibiting SARS-CoV-2 replication and for inhibiting 3CLpro. Masitinib, a broad antiviral, inhibited the main proteases of coronaviruses and picornaviruses and was effective in reducing SARS-CoV-2 replication in mice.
Drayman, Nir↗
Machine Learning-driven Molecular Design for Therapeutic Discovery
The ongoing novel coronavirus pandemic (COVID-19) has highlighted the need for new therapeutics to counter the threat of emerging viral pathogens. The main proteases are a promising target for developing antiviral inhibitors. In this work, we utilized a novel combination of artificial intelligence-driven iterative design of covalent inhibitor candidates, physics-based computational modeling of protein-inhibitor interactions, and “All in One” Native MS biophysical assay screening and characterization of therapeutic candidates. With our existing expertise in hit generation using a particular scaffold as a starting point, we first generated tens of thousands of compounds that preserve the key scaffold. In order to optimize the candidates, we calculated about 136 descriptors consisting of 2D and 3D features for molecules targeting the SARS-CoV-2 Main protease (Mpro). These compounds were initially filtered according to properties and further sorted by predicted binding affinity using our automated docking modeling and machine learning methods. We tested a handful of candidates and identified two as inhibitors of Mpro with micromolar affinities.
59 BASIC BIOLOGICAL SCIENCES↗
Automated AI-driven Molecular Design for Therapeutic Discovery
In recent years, artificial intelligence and machine learning (AI/ML) approaches have revolutionized the process of designing new therapeutics, enabling scientists to rapidly respond to emerging threats from various pathogens. A prime example is the SARS-CoV-2 main protease, a key target for the development of antiviral inhibitors. In this study, we employed a novel, integrated approach that combines AI-driven iterative design of inhibitor candidates, screening based on physio-chemical properties and toxicity, physics-based computational modeling of protein-inhibitor interactions, and AI-assisted analysis of Native MS biophysical assay and characterization of designed candidates. Our deep learning 3D-scaffold model, which uses an input scaffold as a starting point, generated tens of thousands of compounds while preserving the key scaffold. To optimize these candidates, we calculated a comprehensive set of 136 descriptors, including both 2D and 3D molecular features, for compounds targeting the SARS-CoV-2 Main protease (Mpro) and a neurodegenerative disease-associated protein, cyclophilin (Cyp). The generated compounds were initially filtered based on their properties and then ranked according to their predicted binding affinity using our automated modeling and ML methods. Experimental validation of the Mpro candidates showing inhibitory activity demonstrates that our workflow can expedite the therapeutic discovery.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Structural basis for varying drug resistance of SARS-CoV-2 M pro E166 variants
ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) has an essential role in the virus lifecycle and, accordingly, it is a target for antiviral drugs. Multiple studies have identified an M pro mutation (E166V) that confers strong resistance to clinically relevant inhibitors, including nirmatrelvir, but the underlying mechanism is not fully understood. Here, we report on crystal structures of SARS-CoV-2 M pro E166V in complex with nirmatrelvir, ensitrelvir, and bofutrelvir. The structures suggest that resistance is caused in part by the loss of a direct hydrogen bond and also, especially for nirmatrelvir, by a steric clash with the substituted valine residue. In comparison, the binding of bofutrelvir shows greater flexibility, which may help alleviate this steric effect and allow bofutrelvir to fit the mutant active site despite the loss of a direct polar contact. Thermal stability analyses also corroborate E166V most severely affecting the binding of nirmatrelvir and, to lesser and different extents, ensitrelvir and bofutrelvir. We further show that E166I causes even more severe nirmatrelvir resistance, whereas E166A and E166L have much milder effects. These studies shed light on the molecular mechanisms of a key M pro drug resistance mutation and may help inform the design of next-generation inhibitors. IMPORTANCE Using a combination of high-resolution X-ray crystallographic and biochemical analyses, we reveal the molecular mechanisms by which a mutation in the severe acute respiratory syndrome coronavirus 2 main protease (M pro ) confers strong resistance against clinically relevant antiviral drugs that inhibit M pro activity. The results presented here may help inform the design of next-generation inhibitors to combat the problem of therapy resistance.
Microbiology↗
Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV)
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. Here, we describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CL pro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 μM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1e and 1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.
3-Chymotrypsin-like protease (3CLpro)↗
Room-temperature X-ray crystallography reveals the oxidation and reactivity of cysteine residues in SARS-CoV-2 3CL Mpro: insights into enzyme mechanism and drug design
The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL M pro , is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL M pro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N -ethylmaleimide. It is suggested that the zwitterionic Cys145–His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid–base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL M pro inhibitors.
3CL Mpro↗
Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline
A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC 50 values below 10 μM, while candesartan cilexetil had an IC 50 value of approximately 67 µM against M pro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC 50 ) value was the only one greater than the limit of the assay (>100 μM).
59 BASIC BIOLOGICAL SCIENCES↗
A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells
The main protease enzyme (Mpro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that Mpro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported. To enable work on Mpro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) Mpro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing Mpro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with Mpro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that requires conserved residues within the dimerization interface. Proteolytic activity is dispensable for dimerization and, uniquely, a C145A catalytically inactive mutant exhibits enhanced dimerization signal likely due to lower cytotoxicity. Mpro enzymes from multiple different coronaviruses also dimerize in this system, indicating mechanistic conservation. Interestingly, this dimerization biosensor also provides a quantitative read-out of inhibitor-facilitated dimerization. Covalent SARS2 Mpro inhibitors such as nirmatrelvir cause a 3- to 5-fold increase in luciferase activity. Together with corroborating structural, biophysical, and molecular dynamics experiments, our studies support a model in which covalent Mpro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.
SARS-CoV-2 main protease (Mpro/3CLpro)↗
AI-Accelerated Design of Targeted Covalent Inhibitors for SARS-CoV-2
Direct-acting antivirals for the treatment of the COVID-19 pandemic caused by the SARS-CoV-2 virus are needed to complement vaccination efforts. Given the ongoing emergence of new variants, automated experimentation, and active learning based fast workflows for antiviral lead discovery remain critical to our ability to address the pandemic’s evolution in a timely manner. While several such pipelines have been introduced to discover candidates with noncovalent interactions with the main protease (M pro ), here we developed a closed-loop artificial intelligence pipeline to design electrophilic warhead-based covalent candidates. Here, this work introduces a deep learning-assisted automated computational workflow to introduce linkers and an electrophilic “warhead” to design covalent candidates and incorporates cutting-edge experimental techniques for validation. Using this process, promising candidates in the library were screened, and several potential hits were identified and tested experimentally using native mass spectrometry and fluorescence resonance energy transfer (FRET)-based screening assays. We identified four chloroacetamide-based covalent inhibitors of M pro with micromolar affinities (K I of 5.27 μM) using our pipeline. Experimentally resolved binding modes for each compound were determined using room-temperature X-ray crystallography, which is consistent with the predicted poses. The induced conformational changes based on molecular dynamics simulations further suggest that the dynamics may be an important factor to further improve selectivity, thereby effectively lowering KI and reducing toxicity. These results demonstrate the utility of our modular and data-driven approach for potent and selective covalent inhibitor discovery and provide a platform to apply it to other emerging targets.
60 APPLIED LIFE SCIENCES↗
SARS-CoV-2 M pro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals
The main protease of SARS-CoV-2 (M pro ) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in M pro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 M pro mutants provided structural insight into their altered functionality. Additionally, we show M pro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting M pro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Identification and Exploration of a Series of SARS-Cov-2 M Pro Cyano-Based Inhibitors Revealing Ortho-Substitution Effects within the P3 Biphenyl Group
Starting from a simple scaffold hopping exercise based on our previous exploration of cysteine protease inhibitors against legumain, compound 6a was identified as a starting point for the development of a SARS-CoV-2 main protease (M Pro ) inhibitor. Compound 6a displayed submicromolar biochemical potency in the ultrasensitive assay developed by Drag and coworkers. Through an iterative structure−activity relationship campaign, we discovered an unexpected improvement in both biochemical and cellular potency through the incorporation of an ortho substituent within the P3 benzamide. X-ray crystallography revealed that incorporation of the ortho substituent caused a subtle but important binding enhancement of the P1 glutamate group within the M Pro S1 pocket. While incorporation of the ortho substituent improved the potency, the off-target selectivity against a panel of cysteine proteases and cell activity remained suboptimal. Further scanning of the P2 core revealed that incorporation of the 3.1.0 proline could address these issues and afford compound 22e, a highly potent and cellularly active M Pro inhibitor.
COVID-19 infection↗
Dynamic Profiling of β-Coronavirus 3CL Mpro Protease Ligand-Binding Sites
β-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand–protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across β-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.
59 BASIC BIOLOGICAL SCIENCES↗
Development of the Safe and Broad‐Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ‐AA Peptide Scaffold
Abstract SARS‐CoV‐2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (M pro ) is essential in the replication of SARS‐CoV‐2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics‐constrained α, γ‐AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the M pro of SARS‐CoV‐2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET‐based M pro enzymatic assay not only for the M pro of SARS‐CoV‐2 but also for SARS‐CoV and MERS‐CoV, along with HCoVs like HCoV‐OC43, HCoV‐229E, HCoV‐NL63 and HKU1. The X‐ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS‐CoV‐2. Overall, the results suggest that α, γ‐AA peptide could be a promising molecular scaffold in designing novel M pro inhibitors of SARS‐CoV‐2 and other coronaviruses.
Chemistry↗
Exploration of Nirmatrelvir Derivatives as Optimized SARS‐CoV‐2 Antivirals
Nirmatrelvir (NMV) is a SARS‐CoV‐2 antiviral component of the approved COVID‐19 therapeutic Paxlovid. It is a reversible covalent inhibitor of SARS‐CoV‐2 main protease (M Pro ) that is effluxed from human cells by P‐glycoprotein (P‐gp). To identify NMV analogs with improved potency and reduced P‐gp efflux, a structure–activity relationship campaign was conducted. Warheads alternative to nitrile for engaging the active site cysteine were tested showing aldehyde and dichloroacetamide with better enzyme inhibition potency. Crystal structure of MPI‐136−M Pro shows its aldehyde warhead forming a thiohemiacetal with active Cys145 of M Pro . Several S4 binders were explored revealing that an O‐to‐S shift at the N ‐terminal amide leads to better enzyme inhibition. By exploring different combinations of S2, S3, and S4 binders, two inhibitors with better enzyme inhibition potency than NMV were found. Crystal structure of MPI‐148, with ( S )‐2‐azaspiro[4,5]decane‐3‐carboxylate as an alternative S2 binder, shows extensive hydrogen‐bond networks for locking the inhibitor in active site, explaining high affinity of NMV analogs. Further characterization of cellular M Pro engagement and antiviral potency against SARS‐CoV‐2 revealed four inhibitors with greater potency than NMV in P‐gp‐expressing cells. Studies with the P‐gp inhibitor CP‐100356 showed that these compounds were less sensitive to P‐gp inhibition than NMV, consistent with reduced P‐gp‐mediated efflux.
Alugubelli, Yugendar R. [Texas A&M Drug Discovery ↗
Protein data bank: From two epidemics to the global pandemic to mRNA vaccines and Paxlovid
Structural biologists and the open-access Protein Data Bank (PDB) played decisive roles in combating the COVID-19 pandemic. Global biostructure data were turned into global knowledge, allowing scientists and engineers to understand the inner workings of coronaviruses and develop effective countermeasures. Two mRNA vaccines, initially designed with guidance from PDB structures of the SARS-CoV-1 and MERS-CoV spike proteins, prevented infections entirely or reduced the likelihood of morbidity and mortality for more than five billion individual recipients worldwide. Structure-guided drug discovery by Pfizer, Inc (facilitated by PDB structures), initiated in the 2000s in response to SARS-CoV-1 and resumed in 2020, yielded nirmatrelvir (the active ingredient of Paxlovid) -- a potent, orally-bioavailable inhibitor of the SARS-CoV-2 main protease. You've got to love the Protein Data Bank!
Burley, Stephen K.↗