Structure–function relationship of Chikungunya nsP2 protease: A comparative study with papain

Chikungunya virus is a growing human pathogen transmitted by mosquito bite. It causes fever, chills, nausea, vomiting, joint pain, headache, and swelling in the joints. Its replication and propagation depend on the protease activity of the Chikungunya virus‐nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into individual functional units. The N‐terminal segment of papain is structurally identical with the Chikungunya virus‐nsP2 protease. Hence, molecular dynamics simulations were performed to compare molecular mechanism of these proteases. The Chikungunya virus‐snP2 protease shows more conformational changes and adopts an alternate conformation. However, N‐terminal segment of these two proteases has identical active site scaffold with the conserved catalytic diad. Hence, some of the non‐peptide inhibitors of papain were used for induced fit docking at the active site of the nsP2 to assess the binding mode. In addition, the peptides that connect different domains/protein in Chikungunya virus poly‐protein were also subjected for docking. The overall results suggest that the active site scaffold is the same in both the proteases and a possibility exists to experimentally assess the efficacy of some of the papain inhibitors to inhibit the Chikungunya virus‐nsP2.     

Discovery of Novel Peptidomimetics as Irreversible CHIKV NsP2 Protease Inhibitors Using Quantum Mechanical‐Based Ligand Descriptors

Chikungunya virus (CHIKV) is a mosquito‐borne alphavirus. Recent outbreaks of CHIKV infections have been reported in Asia, Africa, and Europe. The symptoms of CHIKV infection include fever, headache, nausea, vomiting, myalgia, rash, and chronic persistent arthralgia. To date, no vaccines or selective antiviral drugs against this important emerging virus have been reported. In this study, the design, synthesis, and antiviral activity screening of new topographical peptidomimetics revealed three potential prototype agents 3a , 4b, and 5d showing 93–100% maximum inhibition of CHIKV replication in cell‐based assay having EC₉₀ of 8.76–9.57 μg/mL. Intensive molecular modeling studies including covalent docking, lowest unoccupied molecular orbital energies, and the atomic condensed Fukui functions calculations strongly suggested the covalent binding of peptidomimetics 3a , 4b, and 5d to CHIKV nsP2 protease leading to permanent enzyme inactivation via Michael adduct formation between α/β‐unsaturated ketone functionality in our designed peptidomimetics and active site catalytic cysteine1013. Furthermore, small molecular weight peptidomimetics 3a and 4b satisfied the Lipinski rule of five for drug‐likeness and showed promising intestinal absorption and aqueous solubility via computational admet studies making them promising hits for further optimization.     

Marine alkaloid oroidin analogues with antiviral potential: A novel class of synthetic compounds targeting the cellular chaperone Hsp90

Marine organisms and their metabolites are a diverse source of scaffolds for potential pharmacological molecular probes and, less frequently, for pharmaceutical lead compounds. In this study, 157 synthetic analogues of marine sponge‐derived alkaloids clathrodin and oroidin were screened against replicon models of two RNA viruses, hepatitis C (HCV) and Chikungunya virus (CHIKV) as part of a larger screening project. Four compounds were found to selectively inhibit the HCV replicon (IC₅₀ 1.6–4.6 μm ). These belong to the 4,5,6,7‐tetrahydrobenzo[1,2‐d]thiazole class of compounds originally designed to target the ATP‐binding site of bacterial DNA gyrase. The ATP‐binding site of this bacterial protein has high structural similarity to the ATP‐binding site of heat‐shock protein 90 (Hsp90), a host cell chaperone universally required for viral replication, which led us to examine inhibition of Hsp90 as the compounds’ potential mechanism of action. Binding of the four hit compounds to Hsp90 was evaluated through microscale thermophoresis and molecular modeling, which supported our hypothesis of interaction with Hsp90 (K_d 18–79 μm ) as basis for the compounds’ antiviral activity. The presented novel structural class of small molecules that target the Hsp90 ATP‐binding site has excellent potential for further antiviral drug development because of the compounds’ low toxicity and synthetic accessibility.     

Molecular modeling study on the resistance mechanism of HCV NS3/4A serine protease mutants R155K, A156V and D168A to TMC435

Hepatitis C virus (HCV) NS3/4A protease represents an attractive drug target for antiviral therapy. However, drug resistance often occurs, making many protease inhibitors ineffective and allowing viral replication to occur. Herein, based on the recently determined structure of NS3/4A-TMC435 complex, atomic-level models of the key residue mutated (R155K, A156V and D168A) NS3/4A-TMC435 complexes were constructed. Subsequently, by using molecular dynamics simulations, binding free energy calculation and substrate envelope analysis, the structural and energetic changes responsible for drug resistance were investigated. The values of the calculated binding free energy follow consistently the order of the experimental activities. More importantly, the computational results demonstrate that R155K and D168A mutations break the intermolecular salt bridges network at the extended S2 subsite and affect the TMC435 binding, while A156V mutation leads to a significant steric clash with TMC435 and further disrupts the two canonical substrate-like intermolecular hydrogen bond interactions (TMC435(N1-H46)?Arg155(O) and Ala157(N-H)?TMC435(O2)). In addition, by structural analysis, all the three key residue mutations occur outside the substrate envelope and selectively weaken TMC435’s binding affinity without effect on its natural substrate peptide (4B5A). These findings could provide some insights into the resistance mechanism of NS3/4A protease mutants to TMC435 and would be critical for the development of novel inhibitors that are less susceptible to drug resistance.     

Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 71 3C and SARS-CoV-2 main protease

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3C^pro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (M^pro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of M^pro to inhibit its activity. We demonstrate that targeting the active site cysteine of M^pro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.     

Molecular docking studies with rabies virus glycoprotein to design viral therapeutics

The genome of rabies virus encodes five proteins; the nucleoprotein, the phosphoprotein, the matrix protein, the glycoprotein, and the RNA-dependent RNA polymerase. Among these, the glycoprotein is the most important as it is the major contributor to pathogenicity and virus neutralizing antibody response. Keeping in mind that glycoprotein is the only protein exposed on the surface of virus and is thought to be responsible for the interaction with the cell membrane, it was attempted to target glycoprotein by a ligand polyethylene glycol 4000, which blocks its active site, as seen by molecular operating environment software, so that it may be possible to prevent the spread of virus into the host. The ligand polyethylene glycol 4000 was retrieved from Research Collaboratory for Structural Bioinformatics protein data bank by providing the glycoprotein sequence to the databank. In this study it was observed that the ligand was successfully docked on a major portion of antigenic site II of glycoprotein by mimicking the virus neutralizing antibodies. This knowledge may be important for the development of novel therapies for the treatment of rabies and other viral diseases in the future.     

Tritiated D-ala1-peptide T binding: A pharmacologic basis for the design of drugs which inhibit HIV receptor binding

The HIV virus initiates its infectious cycle through a high-affinity binding interaction between the envelope protein gp 120 and its receptor, the T4 (or CD4) molecule. An octapeptide sequence, termed peptide T, present in the second variable region of gp 120 from the ARV isolate, has been implicated as the attachement site. The core peptide required for activity has been further refined to a pentapeptide, and homologous pentapeptides are similarly positioned in 21 other sequenced HIV isolates. Utilizing a novel direct binding assay of ³H-D-ala¹-peptide T, we now report that synthetic peptides derived from these other isolates are potent competitors of peptide T binding to T4-positive lymphocytes and brain membranes. Direct peptide T binding is also competable by purified virion gp 120, indicating that these ligands are interactive at the same receptor. Peptide T has sequence relatedness to the peptide vasoactive intestinal peptide (VIP), and VIP and its relevant homologous pentapeptide, VIP[7–11], are also potent inhibitors of peptide T binding. To determine the essential structural features responsible for receptor activity we have studied a series of synthetic peptides substituted with single D-amino acid residues. These data reveal that the tyrosine of position 7 in peptide T, present in all natural viral isolates, is obligate for receptor activity. Structure/function analysis for a large number of analogs is presented. Significantly, this binding assay is highly correlated with peptide bioactivity in several independent systems, indicating that this methodology can be used for rapid screening of novel, potential anti-AIDS therapeutics whose target is inhibition of virus gpl20 binding.     

Structure‐guided Discovery of a Novel Non‐peptide Inhibitor of Dengue Virus NS2B–NS3 Protease

Dengue fever is a fast emerging epidemic‐prone viral disease caused by dengue virus serotypes 1‐4. NS2B–NS3 protease of dengue virus is a validated target to develop antiviral agents. A major limitation in developing dengue virus protease inhibitors has been the lack of or poor cellular activity. In this work, we extracted and refined a pharmacophore model based on X‐ray crystal structure and predicted binding patterns, followed by a three‐dimensional flexible database filtration. These output molecules were screened according to a docking‐based protocol, leading to the discovery of a compound with novel scaffold and good cell‐based bioactivity that has potential to be further optimized. The discovery of this novel scaffold by combination of in silico methods suggests that structure‐guided drug discovery can lead to the development of potent dengue virus protease inhibitors.     

Structural Insights into Plasticity and Discovery of Remdesivir Metabolite GS-441524 Binding in SARS-CoV-2 Macrodomain

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in the host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus targeting this protein domain may offer a therapeutic strategy to combat current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally occurring nucleotides, small molecules, and nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and provide chemical starting points for future inhibitor development.     

Bovine lactoferrin prevents the entry and intercellular spread of herpes simplex virus type 1 in Green Monkey Kidney cells

Lactoferrin (Lf) is a multifunctional glycoprotein that plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Bovine lactoferrin (bLf) has been recognized as a potent inhibitor of human herpetic viruses, such as cytomegalovirus and herpes simplex virus type 1 and 2. BLf has been found to prevent viral infection by binding to heparan sulphate containing proteoglycans that also act as cell receptors for herpetic viruses. In this study we further investigated the inhibiting activity of bLf against herpes simplex virus type 1 (HSV-1) in Green Monkey Kidney (GMK) cells and found that, in addition to the viral adsorption step, bLf also targets the HSV-1 entry process and cell-to-cell viral spread. Our study showed that the inhibition of HSV-1 infectivity by bLf is dependent on its interaction with specific structural viral proteins. Apart from the prevention of early phases of viral infection, cell-to-cell spread inhibition activity of HSV-1 by bLf confirmed that this protein is an outstanding candidate for the treatment of herpetic infections since it would offer the advantage to prevent also viral infections caused by cell-associated virus.     

Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease

The hepatitis C virus (HCV) NS3.4A protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of potent and selective small-molecule inhibitors of HCV NS3.4A protease as oral drug candidates has been hampered by the shallow substrate-binding groove of the protease. Serine trap warheads have been used to covalently anchor inhibitor scaffolds and to increase their affinity to the protease. This review will examine the evolution of covalent inhibitors of the HCV NS3.4A protease from early aldehyde molecules to alpha-ketoamide inhibitors. Kinetic and structural studies of alpha-ketoacid and alpha-ketoamide inhibitors revealed an unusual mechanism of binding in the catalytic site. Optimization of alpha-ketoamide scaffolds by scientists at Vertex and Eli Lilly led to the discovery of VX-950, a novel, potent, selective inhibitor of HCV NS3.4A protease. VX-950 possesses excellent antiviral activity in both HCV replicon cells and human fetal hepatocytes infected with HCV-positive patient sera. In addition, VX-950 exhibits a favorable pharmacokinetic profile in several animal species and demonstrates potent inhibition of the HCV NS3.4A protease activity in a mouse model. In a recent phase 1b clinical trial, VX-950 was able to rapidly reduce the plasma viral load of patients chronically infected with genotype 1 HCV by a mean approximately 3 log(10) in 2 days. The median viral load reduction was 4.4 log(10) for the best dose group after 14 days of dosing. The pre-clinical profile and early clinical data of VX-950 will be discussed in this review.     

Design and synthesis of potent HIV-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity P(2) ligands: structure-activity studies and biological evaluation

The design, synthesis, and evaluation of a new series of hexahydrofuropyranol-derived HIV-1 protease inhibitors are described. We have designed a stereochemically defined hexahydrofuropyranol-derived urethane as the P2-ligand. The current ligand is designed based upon the X-ray structure of 1a-bound HIV-1 protease. The synthesis of (3aS,4S,7aR)-hexahydro-2H-furo[2,3-b]pyran-4-ol, (-)-7, was carried out in optically active form. Incorporation of this ligand provided inhibitor 35a, which has shown excellent enzyme inhibitory activity and antiviral potency. Our structure-activity studies have indicated that the stereochemistry and the position of oxygens in the ligand are important to the observed potency of the inhibitor. Inhibitor 35a has maintained excellent potency against multidrug-resistant HIV-1 variants. An active site model of 35a was created based upon the X-ray structure of 1b-bound HIV-1 protease. The model offers molecular insights regarding ligand-binding site interactions of the hexahydrofuropyranol-derived novel P2-ligand.     

Elucidating the inhibiting mode of AHPBA derivatives against HIV-1 protease and building predictive 3D-QSAR models.

The Lamarckian genetic algorithm of AutoDock 3.0 has been used to dock 27 3(S)-amino-2(S)-hydroxyl-4-phenylbutanoic acids (AHPBAs) into the active site of HIV-1 protease (HIVPR). The binding mode was demonstrated in the aspects of the inhibitor's conformation, subsite interaction, and hydrogen bonding. The data of geometrical parameters (tau(1), tau(2), and tau(3) listed in Table 2) and root mean square deviation values as compared with the known inhibitor, kni272,(28) show that both kinds of inhibitors interact with HIVPR in a very similar way. The r(2) value of 0.860 indicates that the calculated binding free energies correlate well with the inhibitory activities. The structural and energetic differences in inhibitory potencies of AHPBAs were reasonably explored. Using the binding conformations of AHPBAs, consistent and highly predictive 3D-QSAR models were developed by performing CoMFA, CoMSIA, and HQSAR analyses. The reasonable r(corss)(2) values were 0.613, 0.530, and 0.717 for CoMFA, CoMSIA, and HQSAR models, respectively. The predictive ability of these models was validated by kni272 and a set of nine compounds that were not included in the training set. Mapping these models back to the topology of the active site of HIVPR leads to a better understanding of vital AHPBA-HIVPR interactions. Structural-based investigations and the final 3D-QSAR results provide clear guidelines and accurate activity predictions for novel HIVPR inhibitors.     

Identification of irreversible protein splicing inhibitors as potential anti-TB drugs: insight from hybrid non-covalent/covalent docking virtual screening and molecular dynamics simulations

Tuberculosis is responsible for ~3 million deaths annually and is one of the most prevalent infectious diseases known to mankind. Despite ongoing developments in medicine, the emergence of drug resistant Mycobacterium tuberculosis remains of great interest, specifically in developing countries where medical treatment is not readily available. The aim of this study is to identify and explore the binding affinities of novel potential inhibitors that can irreversibly inhibit the intein protein via a covalent bond formation with its active site cysteine. Our search for new leads as potential protein splicing inhibitors is based on Michael acceptor-like structures since they are strong electrophiles which react covalently with the nucleophilic cysteine SH group in the enzyme active site. Structure-based virtual screening using a hybrid non-covalent/covalent docking was performed. Furthermore, molecular dynamic simulations (MD) and extensive post-dynamic analysis were performed in order to ensure the stability of the docked ligand-enzyme complexes and provide insight into the binding affinities and interaction patterns of the screened inhibitors. Interestingly, three novel hits have shown better binding affinity in comparison to experimentally determined compounds with known protein splicing inhibitory activity. MD simulations also revealed that the docked compounds are fairly stable in the protein active site. Per-residue interaction analysis has highlighted the most important active site residues contributing to the inhibitor binding.     

Structure-activity relationship of small-sized HIV protease inhibitors containing allophenylnorstatine.

We designed and synthesized a new class of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing a unique unnatural amino acid, allophenylnorstatine [Apns; (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid], with a hydroxymethylcarbonyl (HMC) isostere as the active moiety. A systematic evaluation of structure-activity relationships for HIV protease inhibition, anti-HIV activities, and pharmacokinetic profiles has led to the delineation of a set of structural charateristics that appear to afford an orally available HIV protease inhibitor. Optimum structures, exemplified by 21f (JE-2147), incorporated 3-hydroxy-2-methylbenzoyl groups as the P2 ligand, (R)-5,5-dimethyl-1,3-thiazolidine-4-carbonyl (Dmt) residue at the P1' site, and 2-methylbenzylcarboxamide group as the P2' ligand. The present study demonstrated that JE-2147 has potent antiviral activities in vitro and exhibits good oral bioavailability and plasma pharmacokinetic profiles in two species of laboratory animals.     

West Nile Virus NS2B/NS3 protease as an antiviral target

West Nile Virus (WNV) has spread rapidly during the last decade across five continents causing disease and fatalities in humans and mammals. It highlights the serious threat to both our health and the economy posed by viruses crossing species, in this case from migratory birds via mosquitoes to mammals. There is no vaccine or antiviral drug for treating WNV infection. One attractive target for antiviral development is a viral trypsin-like serine protease, encoded by the N-terminal 184 amino acids of NS3, which is only active when tethered to its cofactor, NS2B. This protease, NS2B/NS3pro, cleaves the viral polyprotein to release structural and non-structural viral proteins that are essential in viral replication and assembly of new virus particles. Disruption of this protease activity is lethal for virus replication. The NS3 protein also has other enzymes within its sequence (helicase, nucleoside triphosphatase, RNA triphosphatase), all of which are tightly regulated through localisation within membranous compartments in the infected cell. This review describes the various roles of NS3, focussing on NS2B-NS3 protease and its function and regulation in WNV replication and infection. Current advances towards development of antiviral inhibitors of NS2B/NS3pro are examined along with obstacles to their development as an antiviral therapy.     

HIV-1 integrase inhibition: binding sites,structure activity relationships and future perspectives

The integrase enzyme encoded by the human immunodeficiency virus plays an integral role in the viral life cycle, but is as yet unexploited as a clinical drug target. Integrase processes the viral DNA in the cytoplasm, translocates to the nucleus, and catalyzes viral DNA insertion into the host genome. A wide variety of chemical structures inhibit integrase in vitro, yet few of these apparently promising compounds have demonstrated similar efficacy in vivo. Multiple binding targets have been identified for different integrase inhibitors. These targets include the integrase enzyme prior to substrate binding, the viral DNA substrate, and the preintegration complex consisting of oligomeric integrase and the viral DNA. Some known inhibitors are effective only in the presence of divalent manganese as the active site metal ion cofactor, whereas others do not discriminate between manganese and magnesium ions. Integrase inhibition in response to ligand binding at one of multiple sites renders derivation of a simple set of structure activity relationships challenging. Progress toward this goal is reviewed in the context of experimental and theoretical structural information about integrase.