Exploring the evolutionary trajectory and functional landscape of cannabinoid receptors: A comprehensive bioinformatic analysis.

The bioinformatic analysis of cannabinoid receptors (CBRs) CB1 and CB2 reveals a detailed picture of their structure, evolution, and physiological significance within the endocannabinoid system (ECS). The study highlights the evolutionary conservation of these receptors evidenced by sequence alignments across diverse species including humans, amphibians, and fish. Both CBRs share a structural hallmark of seven transmembrane (TM) helices, characteristic of class A G-protein-coupled receptors (GPCRs), which are critical for their signalling functions. The study reports a similarity of 44.58 % between both CBR sequences, which suggests that while their evolutionary paths and physiological roles may differ, there is considerable conservation in their structures. Pathway databases like KEGG, Reactome, and WikiPathways were employed to determine the involvement of the receptors in various signalling pathways. The pathway analyses integrated within this study offer a detailed view of the CBRs interactions within a complex network of cannabinoid-related signalling pathways. High-resolution crystal structures (PDB ID: 5U09 for CB1 and 5ZTY for CB2) provided accurate structural information, showing the binding pocket volume and surface area of the receptors, essential for ligand interaction. The comparison between these receptors' natural sequences and their engineered pseudo-CBRs (p-CBRs) showed a high degree of sequence identity, confirming the validity of using p-CBRs in receptor-ligand interaction studies. This comprehensive analysis enhances the understanding of the structural and functional dynamics of cannabinoid receptors, highlighting their physiological roles and their potential as therapeutic targets within the ECS.

Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir.

AIDS is one of the deadliest diseases in the history of humankind caused by HIV. Despite the technological development, curtailing the viral infection inside human host still remains a challenge. Therapies such as HAART uses a combination of drugs to inhibit the viral activity. One of the important targets includes HIV protease and inhibiting its activity will minimize the production of mature structural proteins. However, the genetic diversity and the occurrence of drug resistant mutations adds complexity to effective drug design. In this study, we aimed at understanding the drug binding mechanism of one such subtype, namely subtype C and its insertion variant L38HL. We performed multiple molecular dynamics simulations along with binding free energy analysis of wild-type and L38HL bound to Atazanavir (ATV). From the analysis, we revealed that the insertion alters the hydrogen bond and hydrophobic interaction networks. The alterations in the interaction networks increase flexibility at the hinge-fulcrum interface. Further, the effects of these changes affect flap tip curling. Moreover, the changes in the hinge-fulcrum-cantilever interface alters the concerted motion of the functional regions leading to change in the direction of flap movement thus causing a subtle change in the active site volume. Additionally, formation of intramolecular hydrogen bonds in the ATV docked to L38HL restricted the movement of R1 and R2 groups thereby altering the interactions. Overall, the changes in the flexibility of flap together with the changes in the active site volume and compactness of the ligand provide insights for increased binding affinity of ATV with L38HL.

Obtaining high yield recombinant Enterococcus faecium nicotinate nucleotide adenylyltransferase for X-ray crystallography and biophysical studies.

Nicotinate nucleotide adenylyltransferase (NNAT) has been a significant research focus on druggable targets, given its indispensability in the biosynthesis of NAD+, which is crucial to the survival of bacterial pathogens. However, no information is available on the structure-function of Enterococcus faecium NNAT (EfNNAT). This study established the expression and purification protocol for obtaining a high-yield recombinant EfNNAT using the E. coli expression system and a single-step IMAC purification method. Approximately 101 mg of EfNNAT was obtained per 7.8 g of wet E. coli cells, estimated to be over 98 % pure. We further characterized the biophysical structure and determined the three-dimensional structure of the EfNNAT. Biophysical studies revealed a dimeric protein with a higher α-helical composition. The highly stable protein crystalizes in multiple conditions, yielding high-quality crystals diffracting between 1.78 and 2.80 Å. Two high-resolution crystal structures of EfNNAT in its native and adenine-bound forms were determined at 1.90 Å and 1.82 Å, respectively. The X-ray structures of the EfNNAT revealed the presence of phosphate and sulfate ions occupying and interacting with conserved amino acid residues within the putative substrate binding site, hence providing insight into the probable substrate preference of EfNNAT and, consequently, why EfNNAT may not prefer ß-nicotinamide mononucleotide as a substrate. With the accessibility to high-resolution structures of EfNNAT, further structural evaluation and drug-based screening can be achieved. Hence, we anticipate that this study will provide the basis for the discovery of structure-based inhibitors against this enzyme.

HIV Protease Hinge Region Insertions at Codon 38 Affect Enzyme Kinetics, Conformational Stability and Dynamics.

HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L- 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5 °C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3-4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.

Semi-quantification and Potency Verification of the HIV Protease Inhibitor Based on the Matrix-Capsid Protein Immobilized Nickel (II)/NTA-Tol/Graphene Oxide/SPCE Electrochemical Biosensor.

Human immunodeficiency virus (HIV) causing acquired immune deficiency syndrome (AIDS) is still a global issue. Long-term drug treatment and nonadherence to medication increase the spread of drug-resistant HIV strains. Therefore, the identification of new lead compounds is being investigated and is highly desirable. Nevertheless, a process generally necessitates a significant budget and human resources. In this study, a simple biosensor platform for semi-quantification and verification of the potency of HIV protease inhibitors (PIs) based on electrochemically detecting the cleavage activity of the HIV-1 subtype C-PR (C-SA HIV-1 PR) was proposed. An electrochemical biosensor was fabricated by immobilizing His6-matrix-capsid (H6MA-CA) on the electrode surface via the chelation to Ni2+-nitrilotriacetic acid (NTA) functionalized GO. The functional groups and the characteristics of modified screen-printed carbon electrodes (SPCE) were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). C-SA HIV-1 PR activity and the effect of PIs were validated by recording changes in electrical current signals of the ferri/ferrocyanide redox probe. The detection of PIs, i.e., lopinavir (LPV) and indinavir (IDV), toward the HIV protease was confirmed by the decrease in the current signals in a dose-dependent manner. In addition, our developed biosensor demonstrates the ability to distinguish the potency of two PIs to inhibit C-SA HIV-1 PR activities. We anticipated that this low-cost electrochemical biosensor would increase the efficiency of the lead compound screening process and accelerate the discovery and development of new HIV drugs.

The Unusual Architecture of RNA-Dependent RNA Polymerase (RdRp)'s Catalytic Chamber Provides a Potential Strategy for Combination Therapy against COVID-19.

The unusual and interesting architecture of the catalytic chamber of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) was recently explored using Cryogenic Electron Microscopy (Cryo-EM), which revealed the presence of two distinctive binding cavities within the catalytic chamber. In this report, first, we mapped out and fully characterized the variations between the two binding sites, BS1 and BS2, for significant differences in their amino acid architecture, size, volume, and hydrophobicity. This was followed by investigating the preferential binding of eight antiviral agents to each of the two binding sites, BS1 and BS2, to understand the fundamental factors that govern the preferential binding of each drug to each binding site. Results showed that, in general, hydrophobic drugs, such as remdesivir and sofosbuvir, bind better to both binding sites than relatively less hydrophobic drugs, such as alovudine, molnupiravir, zidovudine, favilavir, and ribavirin. However, suramin, which is a highly hydrophobic drug, unexpectedly showed overall weaker binding affinities in both binding sites when compared to other drugs. This unexpected observation may be attributed to its high binding solvation energy, which disfavors overall binding of suramin in both binding sites. On the other hand, hydrophobic drugs displayed higher binding affinities towards BS1 due to its higher hydrophobic architecture when compared to BS2, while less hydrophobic drugs did not show a significant difference in binding affinities in both binding sites. Analysis of binding energy contributions revealed that the most favorable components are the ΔEele, ΔEvdw, and ΔGgas, whereas ΔGsol was unfavorable. The ΔEele and ΔGgas for hydrophobic drugs were enough to balance the unfavorable ΔGsol, leaving the ΔEvdw to be the most determining factor of the total binding energy. The information presented in this report will provide guidelines for tailoring SARS-CoV-2 inhibitors with enhanced binding profiles.

Understanding Drug Resistance of Wild-Type and L38HL Insertion Mutant of HIV-1 C Protease to Saquinavir.

Acquired immunodeficiency syndrome (AIDS) is one of the most challenging infectious diseases to treat on a global scale. Understanding the mechanisms underlying the development of drug resistance is necessary for novel therapeutics. HIV subtype C is known to harbor mutations at critical positions of HIV aspartic protease compared to HIV subtype B, which affects the binding affinity. Recently, a novel double-insertion mutation at codon 38 (L38HL) was characterized in HIV subtype C protease, whose effects on the interaction with protease inhibitors are hitherto unknown. In this study, the potential of L38HL double-insertion in HIV subtype C protease to induce a drug resistance phenotype towards the protease inhibitor, Saquinavir (SQV), was probed using various computational techniques, such as molecular dynamics simulations, binding free energy calculations, local conformational changes and principal component analysis. The results indicate that the L38HL mutation exhibits an increase in flexibility at the hinge and flap regions with a decrease in the binding affinity of SQV in comparison with wild-type HIV protease C. Further, we observed a wide opening at the binding site in the L38HL variant due to an alteration in flap dynamics, leading to a decrease in interactions with the binding site of the mutant protease. It is supported by an altered direction of motion of flap residues in the L38HL variant compared with the wild-type. These results provide deep insights into understanding the potential drug resistance phenotype in infected individuals.

Non-active site mutations in the HIV protease: Diminished drug binding affinity is achieved through modulating the hydrophobic sliding mechanism.

The global HIV/AIDS epidemic still currently affects approximately 38 million individuals globally. The protease enzyme of the human immunodeficiency virus is a major drug target in antiviral therapy, however, under the influence of reverse transcriptase and in the context of drug pressure, the rapid PR mutation rate contributes significantly to clinical failure. The set of cooperative non-active site mutations, I13V/I62V/V77I, have been associated with reduced inhibitor susceptibility and are the focus of the current study. When compared to the wild-type protease the mutant protease exhibited decreased binding affinities towards ATV and DRV by 64- and 12-fold, respectively, and decreased the overall favourable Gibbs free energy for ATV, DRV, RTV and SQV. Moreover, these mutations decreased the thermal stability of the protease when in complex with ATV and DRV by approximately 6.4 and 4.2 °C, respectively. The crystal structure of the mutant protease revealed that the location of these mutations and their effect on the hydrophobic sliding mechanism may be crucial in their role in resistance.

SARS-CoV-2 Spike Protein Unlikely to Bind to Integrins via the Arg-Gly-Asp (RGD) Motif of the Receptor Binding Domain: Evidence From Structural Analysis and Microscale Accelerated Molecular Dynamics.

The Receptor Binding Domain (RBD) of SARS-CoV-2 virus harbors a sequence of Arg-Gly-Asp tripeptide named RGD motif, which has also been identified in extracellular matrix proteins that bind integrins as well as other disintegrins and viruses. Accordingly, integrins have been proposed as host receptors for SARS-CoV-2. However, given that the microenvironment of the RGD motif imposes a structural hindrance to the protein-protein association, the validity of this hypothesis is still uncertain. Here, we used normal mode analysis, accelerated molecular dynamics microscale simulation, and protein-protein docking to investigate the putative role of RGD motif of SARS-CoV-2 RBD for interacting with integrins. We found, that neither RGD motif nor its microenvironment showed any significant conformational shift in the RBD structure. Highly populated clusters of RBD showed no capability to interact with the RGD binding site in integrins. The free energy landscape revealed that the RGD conformation within RBD could not acquire an optimal geometry to allow the interaction with integrins. In light of these results, and in the event where integrins are confirmed to be host receptors for SARS-CoV-2, we suggest a possible involvement of other residues to stabilize the interaction.

Elasticity-Associated Functionality and Inhibition of the HIV Protease.

HIV protease plays a critical role in the life cycle of the virus through the generation of mature and infectious virions. Detailed knowledge of the structure of the enzyme and its substrate has led to the development of protease inhibitors. However, the development of resistance to all currently available protease inhibitors has contributed greatly to the decreased success of antiretroviral therapy. When therapy failure occurs, multiple mutations are found within the protease sequence starting with primary mutations, which directly impact inhibitor binding, which can also negatively impact viral fitness and replicative capacity by decreasing the binding affinity of the natural substrates to the protease. As such, secondary mutations which are located outside of the active site region accumulate to compensate for the recurrently deleterious effects of primary mutations. However, the resistance mechanism of these secondary mutations is not well understood, but what is known is that these secondary mutations contribute to resistance in one of two ways, either through increasing the energetic penalty associated with bringing the protease into the closed conformation, or, through decreasing the stability of the protein/drug complex in a manner that increases the dissociation rate of the drug, leading to diminished inhibition. As a result, the elasticity of the enzyme-substrate complex has been implicated in the successful recognition and catalysis of the substrates which may be inferred to suggest that the elasticity of the enzyme/drug complex plays a role in resistance. A realistic representation of the dynamic nature of the protease may provide a more powerful tool in structure-based drug design algorithms.

Cantilever-centric mechanism of cooperative non-active site mutations in HIV protease: Implications for flap dynamics.

The HIV-1 protease is an important drug target in antiretroviral therapy due to the crucial role it plays in viral maturation. A greater understanding of the dynamics of the protease as a result of drug-induced mutations has been successfully elucidated using computational models in the past. We performed induced-fit docking studies and molecular dynamics simulations on the wild-type South African HIV-1 subtype C protease and two non-active site mutation-containing protease variants; HP3 PR and HP4 PR. The HP3 PR contained the I13V, I62V, and V77I mutations while HP4 PR contained the same mutations with the addition of the L33F mutation. The simulations were initiated in a cubic cell universe containing explicit solvent, with the protease variants beginning in the fully closed conformation. The trajectory for each simulation totalled 50 ns. The results indicate that the mutations increase the dynamics of the flap, hinge, fulcrum and cantilever regions when compared to the wild-type protease while in complex with protease inhibitors. Specifically, these mutations result in the protease favouring the semi-open conformation when in complex with inhibitors. Moreover, the HP4 PR adopted curled flap tip conformers which coordinated several water molecules into the active site in a manner that may reduce inhibitor binding affinity. The mutations affected the thermodynamic landscape of inhibitor binding as there were fewer observable chemical contacts between the mutated variants and saquinavir, atazanavir and darunavir. These data help to elucidate the biophysical basis for the selection of cooperative non-active site mutations by the HI virus.

Targeting the SARS-CoV-2 main protease using FDA-approved Isavuconazonium, a P2-P3 α-ketoamide derivative and Pentagastrin: An in-silico drug discovery approach.

The SARS-CoV-2 main protease (Mpro) is an attractive target towards discovery of drugs to treat COVID-19 because of its key role in virus replication. The atomic structure of Mpro in complex with an α-ketoamide inhibitor (Lig13b) is available (PDB ID:6Y2G). Using 6Y2G and the prior knowledge that protease inhibitors could eradicate COVID-19, we designed a computational study aimed at identifying FDA-approved drugs that could interact with Mpro. We searched the DrugBank and PubChem for analogs and built a virtual library containing ∼33,000 conformers. Using high-throughput virtual screening and ligand docking, we identified Isavuconazonium, a ketoamide inhibitor (α-KI) and Pentagastrin as the top three molecules (Lig13b as the benchmark) based on docking energy. The ΔGbind of Lig13b, Isavuconazonium, α-KI, Pentagastrin was -28.1, -45.7, -44.7, -34.8 kcal/mol, respectively. Molecular dynamics simulation revealed that these ligands are stable within the Mpro active site. Binding of these ligands is driven by a variety of non-bonded interaction, including polar bonds, H-bonds, van der Waals and salt bridges. The overall conformational dynamics of the complexed-Mpro was slightly altered relative to apo-Mpro. This study demonstrates that three distinct classes molecules, Isavuconazonium (triazole), α-KI (ketoamide) and Pentagastrin (peptide) could serve as potential drugs to treat patients with COVID-19.

Molecular basis of inhibition of Schistosoma japonicum glutathione transferase by ellagic acid: Insights into biophysical and structural studies.

Schistosoma japonicum glutathione transferase (Sj26GST), an enzyme central to detoxification of electrophilic compounds in the parasite, is upregulated in response to drug treatment. Therefore, Sj26GST may serve as a potential therapeutic target for the treatment of schistosomiasis. Herewith, we describe the structural basis of inhibition of Sj26GST by ellagic acid (EA). Using 1-chloro-2,4-dinitrobenzene and reduced glutathione (GSH) as Sj26GST substrates, EA was shown to inhibit Sj26GST activity by 66 % with an IC50 of 2.4 µM. Fluorescence spectroscopy showed that EA altered the polarity of the environment of intrinsic tryptophan and that EA decreased (in a dose-dependent manner) the interaction between Sj26GST and 8-Anilino-1-naphthalenesulfonate (ANS), which is a known GST H-site ligand. Thermodynamic studies indicated that the interaction between Sj26GST and EA is spontaneous (ΔG = -29.88 ± 0.07 kJ/mol), enthalpically-driven (ΔH = -9.48 ± 0.42 kJ/mol) with a favourable entropic change (ΔS = 20.40 ± 0.08 kJ/mol/K), and with a stoichiometry of four EA molecules bound per Sj26GST dimer. The 1.53 Å-resolution Sj26GST crystal structure (P 21 21 21 space group) complexed with GSH and EA shows that EA binds primarily at the dimer interface, stabilised largely by Van der Waal forces and H-bonding. Besides, EA bound near the H-site and less than 3.5 Å from the ε-NH2 of the γ-glutamyl moiety of GSH, in each subunit.

Two-Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli.

Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease-containing inclusion bodies are treated with 8 M urea and purified via cation-exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion-exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Recovery, refolding, and purification of HIV protease from inclusion bodies Support Protocol 1: Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coli Support Protocol 2: Determination of the active site concentration of HIV protease via isothermal titration calorimetry.

Design, synthesis and biological evaluation of novel 2-(5-aryl-1H-imidazol-1-yl) derivatives as potential inhibitors of the HIV-1 Vpu and host BST-2 protein interaction.

Novel ethyl 2-(5-aryl-1H-imidazol-1-yl)-acetates 17 and propionates 18, together with their acetic acid 19 and acetohydrazide 20 derivatives, were designed and synthesized using TosMIC chemistry. Biological evaluation of these newly synthesized scaffolds in the HIV-1 Vpu- Host BST-2 ELISA assay identified seven hits (17a, 17b, 17c, 17g, 18a, 20f and 20g) with greater than 50% inhibitory activity. These hits were validated in the HIV-1 Vpu- Host BST-2 AlphaScreen™ and six of the seven compounds were found to have comparable percentage inhibitory activities to those of the ELISA assay. Compounds 17b and 20g, with consistent percentage inhibitory activities across the two assays, had IC50 values of 11.6 ± 1.1 µM and 17.6 ± 0.9 µM in a dose response AlphaScreen™ assay. In a cell-based HIV-1 antiviral assay, compound 17b exhibited an EC50 = 6.3 ± 0.7 µM at non-toxic concentrations (CC50 = 184.5 ± 0.8 µM), whereas compound 20g displayed antiviral activity roughly equivalent to its toxicity (CC50 = 159.5 ± 0.9 µM). This data suggests that compound 17b, active in both cell-based and biochemical assays, provides a good starting point for the design of possible lead compounds for prevention of HIV-1 Vpu and host BST-2 protein binding in new anti-HIV therapeutics.

Synthesis, Characterization and Biological Activity of Some Dithiourea Derivatives.

Novel dithiourea derivatives have been designed as HIV-1 protease inhibitors using Autodock 4.2, synthesized and characterized by spectroscopic methods and microanalysis. 1-(3-Bromobenzoyl)-3-[2-([(3-bromophenyl)formami-do]methanethioylamino)phenyl]thiourea (10) and 3-benzoyl-1[(phenylformamido)methanethioyl]aminothiourea (12) gave a percentage viability of 17.9 ± 5.6% and 11.2 ± 0.9% against Trypanosoma brucei. Single crystal X-ray dif-fraction analysis of 1-benzoyl-3-(5-methyl-2-[(phenylformamido)methanethioyl]aminophenyl)thiourea (1), 3-ben-zoyl-1-(2-[(phenylformamido)methanethioyl]aminoethyl)thiourea (11), 3-benzoyl-1-[(phenylformamido)methan-ethioyl]aminothiourea (12) and 3-benzoyl-1-(4-[(phenylformamido)methanethioyl]aminobutyl)thiourea (14) have been presented. 1-(3-Bromobenzoyl)-3-[2-([(3-bromophenyl)formamido]methanethioylamino)phenyl]thiourea (10) gave a percentage inhibition of 97.03 ± 0.37% against HIV-1 protease enzyme at a concentration of 100 ?M.

Antimicrobial function of short amidated peptide fragments from the tick-derived OsDef2 defensin.

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3 µM against Escherichia coli. Killing was reached within 10 minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.

Kinetic and thermodynamic characterisation of HIV-protease inhibitors against E35D↑G↑S mutant in the South African HIV-1 subtype C protease.

Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitors against the mutant (p < .0001). Amprenavir and ritonavir showed the least decrease, but still significant reduced activity in comparison to the wildtype (4 and 5 folds, respectively, p = .0021 and .003, respectively). Nelfinavir and atazanavir were the worst inhibitors against the variant as seen from the IC50, with values of 1401 ± 3.0 and 685 ± 3.0 nM, respectively. Thermodynamics data showed less favourable Gibbs free binding energies for the protease inhibitors to the mutant.

Drug susceptibility and replication capacity of a rare HIV-1 subtype C protease hinge region variant.

BACKGROUND: Protease inhibitors form the main component of second-line antiretroviral treatment in South Africa. Despite their efficacy, mutations arising within the HIV-1 gag and protease coding regions contribute to the development of resistance against this class of drug. In this paper we investigate a South African HIV-1 subtype C Gag-protease that contains a hinge region mutation and insertion (N37T↑V). METHODS: In vitro single-cycle drug susceptibility and viral replication capacity assays were performed on W1201i, a wild-type reference isolate (MJ4) and a chimeric construct (MJ4GagN37T↑VPR). Additionally, enzyme assays were performed on the N37T↑V protease and a wild-type reference protease. RESULTS: W1201i showed a small (threefold), but significant (P<0.0001) reduction in drug susceptibility to darunavir compared with MJ4. Substitution of W1201i-Gag with MJ4-Gag resulted in an additional small (twofold), but significant (P<0.01) reduction in susceptibility to lopinavir and atazanavir. The W1201i pseudovirus had a significantly (P<0.01) reduced replication capacity (16.4%) compared with the MJ4. However, this was dramatically increased to 164% (P<0.05) when W1201i-Gag was substituted with MJ4-Gag. Furthermore, the N37T↑V protease displayed reduced catalytic processing compared with the SK154 protease. CONCLUSIONS: Collectively, these data suggest that the N37T↑V mutation and insertion increases viral infectivity and decreases drug susceptibility. These variations are classified as secondary mutations, and indirectly impact inhibitor binding, enzyme fitness and enzyme stability. Additionally, polymorphisms arising in Gag can modify the impact of protease with regards to viral replication and susceptibility to protease inhibitors.

Low Frequency of Protease Inhibitor Resistance Mutations and Insertions in HIV-1 Subtype C Protease Inhibitor-Naïve Sequences.

Human immunodeficiency virus-1 (HIV-1) protease sequences from 2,225 protease inhibitor (PI)-naïve HIV-1 subtype C-infected individuals collected over a 14-year period were analyzed for polymorphisms. Over 50% of sequences differed from an HIV-1 subtype B consensus sequence at 8 of the 99 amino acids at residues 12, 15, 19, 36, 41, 69, 89, and 93, but not in the functionally important regions. The frequency of primary resistance and accessory mutations occurred in <1% of the sequences. Of note, 11 sequences (0.5%) harbored amino acid insertions between residues 36 and 39, located in the elbow of the flap region. The insertions were found throughout the 13-year period. Occurrence of insertions in subtype C viruses is rare and viruses remain sensitive to currently used PIs (lopinavir/r, atazanavir/r, and darunavir/r). However, ongoing characterization of isolates is required to identify changes that may impact PI treatment since PIs are part of standard SA regimens.