Unveiling a Hidden Pocket in HIV-1 Protease: New Insights Into Retroviral Protease Cantilever-Tip Region Characteristics.

The HIV-1 protease is critical for the process of viral maturation and as such, it is one of the most well characterized proteins in the Protein Data Bank. There is some evidence to suggest that the HIV-1 protease is capable of accommodating small molecule fragments at several locations on its surface outside of the active site. However, some pockets on the surface of proteins remain unformed in the apo structure and are termed "cryptic sites." To date, no cryptic sites have been identified in the structure of HIV-1 protease. Here, we characterize a novel cryptic cantilever pocket on the surface of the HIV-1 protease through mixed-solvent molecular dynamics simulations using several probes. Interestingly, we noted that several homologous retroviral proteases exhibit evolutionarily conserved dynamics in the cantilever region and possess a conserved pocket in the cantilever region. Immobilization of the cantilever region of the HIV-1 protease via disulfide cross-linking resulted in curling-in of the flap tips and the propensity for the protease to adopt a semi-open flap conformation. Structure-based analysis and fragment-based screening of the cryptic cantilever pocket suggested that the pocket may be capable of accommodating ligand structures. Furthermore, molecular dynamics simulations of a top scoring fragment bound to the cryptic pocket illustrated altered flap dynamics of the fragment-bound enzyme. Together, these results suggest that the mobility of the cantilever region plays a key role in the global dynamics of retroviral proteases. Therefore, the cryptic cantilever pocket of the HIV-1 protease may represent an interesting target for future in vitro studies.

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.

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.

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility.

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

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.

I36T↑T mutation in South African subtype C (C-SA) HIV-1 protease significantly alters protease-drug interactions.

The efficacy of HIV-1 protease (PR) inhibition therapies is often compromised by the emergence of mutations in the PR molecule that reduces the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for natural substrates. In the present study, we used a recombinant HIV-1 C-SA PR and a recently reported variant for inhibition (Ki, IC50) and thermodynamic studies against nine clinically used inhibitors. This is the first time that binding free energies for C-SA PR and the mutant are reported. This variant PR harbours a mutation and insertion (I36T↑T) at position 36 of the C-SA HIV-1 PR, and did not show a significant difference in the catalytic effect of the HIV-1 PR. However, the nine clinically approved HIV PR drugs used in this study demonstrated weaker inhibition and lower binding affinities toward the variant when compared to the wild type HIV-1 PR. All the protease inhibitors (PIs), except Amprenavir and Ritonavir exhibited a significant decrease in binding affinity (p<0.0001). Darunavir and Nelfinavir exhibited the weakest binding affinity, 155- and 95-fold decreases respectively, toward the variant. Vitality values for the variant PR, against the seven selected PIs, confirm the impact of the mutation and insertion on the South African HIV-1 subtype C PR. This information has important clinical implications for thousands of patients in Sub-Saharan Africa.

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 µmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities.

Non-B HIV-1 subtypes in sub-Saharan Africa: impact of subtype on protease inhibitor efficacy.

In 2012, 25 million people [71% of global human immunodeficiency virus (HIV) infection] were estimated to be living with HIV in sub-Saharan Africa. Of these, approximately 1.6 million were new infections and 1.2 million deaths occurred. South Africa alone accounted for 31% of HIV/acquired immunodeficiency syndrome (AIDS) deaths in sub-Saharan Africa. This disturbing statistic indicates that South Africa remains the epicenter of the HIV/AIDS pandemic, compounded by the fact that only 36% of HIV-positive patients in South Africa have access to antiretroviral (ARV) treatment. Drug resistance mutations have emerged, and current ARVs show reduced efficacy against non-B subtypes. In addition, several recent studies have shown an increased prevalence of non-B African HIV strains in the Americas and Europe. Therefore, the use of ARVs in a non-B HIV-1 subtype context requires further investigation. HIV-1 subtype C protease, found largely in sub-Saharan Africa, has been under-investigated when compared with the subtype B protease, which predominates in North America and Europe. This review, therefore, focuses on HIV-1 proteases from B and C subtypes.

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.

Synthesis and evaluation of coumarin derivatives as potential dual-action HIV-1 protease and reverse transcriptase inhibitors.

Baylis-Hillman-derived 3-(benzylaminomethyl)coumarins have been treated, sequentially, with chloroacetyl chloride and propargylamine to afford alkynylated coumarins as substrates for Click Chemistry reactions with azidothymidine (AZT) in the presence of a Cu(I) catalyst. The dual-action HIV-1 protease (PR) and reverse transcriptase (RT) inhibition potential of the resulting N-benzylated cycloaddition products, and a series of non-benzylated analogues, has been explored using saturation transfer difference (STD) NMR, computer modelling and enzyme inhibition techniques.

Structural and functional characterization of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy-terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os-C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94-15 µg/ml) to both Gram-positive and Gram-negative bacteria, whereas the parent peptide only exhibited Gram-positive antibacterial activity. The Os peptide was found to be two-fold more active than Os-C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os-C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane-mimicking environment, the cysteine-containing peptide has a higher α-helical content. Both peptides were found to be non-toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted.

Synthesis, 2D-NMR and molecular modelling studies of pentacycloundecane lactam-peptides and peptoids as potential HIV-1 wild type C-SA protease inhibitors.

In this study, eight non-natural peptides and peptoids incorporating the pentacycloundecane (PCU) lactam were designed and synthesized as potential inhibitors of the wild type C-SA HIV-protease. Five of these inhibitors gave IC(50) values ranging from 0.5 up to 0.75 µM against the resistance-prone wild type C-South African HIV-protease. NMR EASY-ROESY studies enabled us to describe the secondary structure of three of these compounds in solution. The 3D structures of the selected cage peptides were also modelled in solution using QM/MM/MD simulations. Satisfactory agreement between the NMR observations and the low energy calculated structures exists. Only one of these inhibitors (11 peptoid), which showed the best IC(50)(0.5 µM), exhibited a definable 3-D structure in solution. Autodock4 and AutodockVina were used to model the potential interaction between these inhibitors and the HIV-PR. It appears that the docking results are too crude to be correlated with the relative narrow range of experimental IC(50) values (0.5-10 µM). The PCU-peptides and peptoides were several orders less toxic (145 µM for 11 and 102 µM for 11 peptoid) to human MT-4 cells than lopinavir (0.025 µM). This is the first example of a polycyclic cage framework to be employed as an HIV-PR transition state analogue inhibitor and can potentially be utilized for other diseases related proteases. [Figure: see text].

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.

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.

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.

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.

Euphorbia mauritanica and Kedrostis hirtella extracts can induce anti-proliferative activities in lung cancer cells.

Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 µg/ml in A549 cells over 22 h and at 10 µg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results.