Focus on chirality of HIV-​1 non-​nucleoside reverse transcriptase inhibitors

Chiral HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are of great interest since one enantiomer is often more potent than the corresponding counterpart against the HIV-1 wild type (WT) and the HIV-1 drug resistant mutant strains. This review exemplifies the various studies made to investigate the effect of chirality on the antiretroviral activity of top HIV-1 NNRTI compounds, such as nevirapine (NVP), efavirenz (EFV), alkynyl- and alkenylquinazolinone DuPont compounds (DPC), diarylpyrimidine (DAPY), dihydroalkyloxybenzyloxopyrimidine (DABO), phenethylthiazolylthiourea (PETT), indolylarylsulfone (IAS), arylphosphoindole (API) and trifluoromethylated indole (TFMI) The chiral separation, the enantiosynthesis, along with the biological properties of these HIV-1 NNRTIs, are discussed

Molecular modeling studies on HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90 as a potential anti-HIV-1 target.

Masters Degree. University of KwaZulu-Natal, Durban.Human immunodeficiency virus (HIV) infection is the leading cause of death globally. This dissertation addresses two HIV-1 target proteins namely, HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90. More specifically for HIV-1 RT, a case study for the identification of potential inhibitors as anti-HIV agents was carried out. A more refined virtual screening (VS) approach was implemented, which was an improvement on work previously published by our group- “target-bound pharmacophore modeling approach”. This study generated a pharmacophore library based only on highly contributing amino acid residues (HCAAR), instead of arbitrary pharmacophores, most commonly used in the conventional approaches in literature. HCAAR were distinguished based on free binding energy (FBE) contributions, obtained using calculation from molecular dynamics (MD) simulations. Previous approaches have relied on the docking score (DS) to generate energy-based pharmacophore models. However, DS are reportedly unreliable. Thus we present a model for a per-residue energy decomposition (PRED), constructed from MD simulation ensembles generating a more trustworthy pharmacophore model which can be applied in drug discovery workflow. This approach was employed in screening for potential HIV-1 RT inhibitors using the pharmacophoric features of the compound GSK952. The complex was subjected to docking and thereafter MD simulations confirmed the stability of the system. Experimentally determined inhibitors with known HIV-RT inhibitory activity were used to validate the proposed protocol. Two potential hits ZINC46849657 and ZINC54359621 showed a significant potential with regards to FBE. Reported results obtained from this work confirm that this new approach is favourable to the future of drug design process. Hsp90 was recently discovered to play a vital role in HIV-1 replication. Thus has emerged, as a promising target for anti-HIV-1 drugs. The molecular mechanism of Hsp90 is poorly understood, thus the second study was aimed to address this issue and provide a clear insight to the inhibition mechanism of Hsp90. Reasonable continuous MD simulations were employed for both unbound and bound Hsp90 conformations, to understand the dimerization and inhibition mechanisms. Results demonstrated that coumermycin A1 (C-A1), a newly discovered Hsp90 inhibitor, binds at the CTD dimer of Hsp90 and lead to a significant separation between orthogonally opposed residues, such as Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669A. A Large difference in magnitudes was observed in the radius of gyration (Rg), per-residue fluctuation, root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) confirming a completely more flexible state for the unbound conformation associated with dimerization. Whereas, a less globally correlated motion in the case of the bound conformer of Hsp90 approved a reduction of the dimeric process. This undoubtedly underlines the inhibition process due to ligand binding. The detailed dynamic analyses of Hsp90 presented herein are believed to give a greater insight and understanding to the function and mechanisms of inhibition of Hsp90. The report on the inhibitor-binding mode would also be of great assistance in the design of prospective inhibitors against Hsp90 as potential HIV target

Putative HIV-1 reverse transcriptase inhibitors: design, synthesis, in vitro evaluation and in silico analysis.

Masters Degree. University of KwaZulu-Natal, Durban.One of the most significant treatments for HIV-1 infection has been the combination of drugs targeting the HIV life cycle with the aim of preventing further destruction of the host immune system. This study addresses the design, synthesis, in vitro evaluation, and in silico analysis of putative HIV-1 reverse transcriptase (RT) inhibitors. The inhibitors comprise two structurally diverse components which are intended to bind separately to the enzyme allosteric site and to a location at, or close to, the polymerase active site. Therefore, the hydrophobic N-tritylated phalo-DL-phenylalanine derivatives (fluoro, chloro, bromo, iodo) have been coupled to 8-(6- aminohexyl) amino-adenosine-3',5'-cyclic monophosphate through N-hydroxysuccinimidecarbodiimide chemistry. Compounds were characterized by thin layer chromatography, UV spectroscopy, MALDI-TOF mass spectrometry and proton NMR spectrometry. A reverse transcriptase colorimetric assay kit, which features a sandwich ELISA protocol, based on biotin-avidin and digoxygenin-anti DIG interactions, was used for quantitative determination of the inhibitory effect of synthesized compounds on recombinant HIV-1 reverse transcriptase activity in vitro. Molecular docking simulations of the chimeric inhibitors within the allosteric site of HIV-1 RT, were performed using AutoDock Vina. The predicted binding associations were compared with laboratory findings on HIV-1 RT inhibition. Two dimensional representations of protein-ligand interactions were generated using LigPlot. The non-halogenated N-trityl-L-phenylalanine-8-(6-aminohexyl)amino-adenosine-3',5'-cyclic monophosphate derivative (4a) inhibited RT activity down to 57 % at 10-4 M, while the Ntrityl-para-fluoro-DL-phenylalanine-8-(6-aminohexyl)aminoadenosine-3′,5′-cyclic monophosphate derivative (4b) was the strongest RT inhibitor reducing RT activity to 69 % at 10-7 M (IC50 = 29.2 μM). In the same assay, Nevirapine, a first-line anti-retroviral drug, showed a decline in RT activity down to 43% at 10-5 M (IC50 = 3.03 μM). Ranking of inhibitors according to estimated docking energies obtained from in silico docking was in excellent agreement with potencies calculated from experimental studies. The docking score of N-trityl-para-fluoro-DL-phenylalaline-8-(6-aminohexyl)amino-adenosine-3',5'-cyclic monophosphate was -8.8 kcal/mol, while that of Nevirapine was -9.9 kcal/mol. The benzene rings of the N-trityl-fluoro-DL-phenylalanine-8-(6-aminohexyl) amino-adenosine-3',5'-cyclic monophosphate derivative formed hydrophobic interactions with hydrophobic, non-aromatic amino acid residues Pro176 and Val179 in the allosteric site. Nevirapine, on the other hand showed strong van der Waals interactions with Val106 ,Val179 and Tyr188 due to the aromatic properties of the pyridine ring. Possible π-π stacking between phenyl rings of Nevirapine and Tyr 181/Tyr188 aromatic side chains may also be present. Other HIV-1 RT large subunit residues in the allosteric site common to the binding of Nevirapine and the active para-fluoro derivative include Lys101, Tyr318, Leu 100, Trp229 and Phe227. Apparent binding to the allosteric site suggests that compounds may be acting primarily as non-nucleoside reverse transcriptase inhibitors (NNRTIs)

Ribonuclease H/DNA polymerase HIV-1 reverse transcriptase dual inhibitor: mechanistic studies on the allosteric mode of action of isatin-based compound RMNC6

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 R

Computational development of rubromycin-based lead compounds for HIV-1 reverse transcriptase inhibition

The binding of several rubromycin-based ligands to HIV1-reverse transcriptase was analyzed using molecular docking and molecular dynamics simulations. MM-PBSA analysis and examination of the trajectories allowed the identification of several promising compounds with predicted high affinity towards reverse transcriptase mutants which have proven resistant to current drugs. Important insights on the complex interplay of factors determining the ability of ligands to selectively target each mutant have been obtained

Molecular dynamics simulation of drug resistance in HIV-1 protease and reverse transcriptase

The emergence of drug resistant strains of HIV represents a major challenge in the treatment of patients who contract the virus. We investigate the use of classical molecular dynamics to give quantitative and qualitative molecular insight into the causes of resistance in the two main drug targets in HIV, protease and reverse transcriptase. We initially establish a simulation and free energy analysis protocol for the study of resistance in protease. Focusing on the binding of the inhibitor lopinavir to a series of six mutants with increasing resistance we demonstrate that ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations. We achieve accurate and converged relative binding free energies, reproducible to within 0.5 kcal mol^-1. The experimentally derived ranking of the systems is reproduced with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal mol^-1. Our protocol is then applied to investigate a patient derived viral sequence for which contradictory resistance assessments for lopinavir were obtained from existing clinical decision support systems (CDSS). Mutations at only three locations (L10I, A71I/V and L90M) in uenced the ranking. Free energies were computed for HXB2 wildtype sequences incorporating each mutation individually and all possible combinations, along with the full patient sequence. Only in the case of the patient sequence was any resistance observed. This observation suggests an explanation for the discordance found using the CDSS. The effects on drug binding of the mutations at positions 10, 71 and 90 appear to be highly dependent on the background mutations present in the remainder of the sequence. In preparation for the extension of our simulation and free energy protocol to reverse transcriptase the impact of binding both natural DNA substrates and two non nucleoside reverse transcriptase inhibitor (NNRTI) class drugs on the dynamics of reverse transcriptase are investigated. Free energies of both inhibitors (efavirenz and neviripine) are determined which are seen to be independent of the subdomain motions of the protein observed during simulation. Preliminary calculations of the free energies for a set of NNRTI resistant mutants bound to efavirenz are also presented

Development of Glucan Particle Encapsulated Nevirapine as a Topical HIV Microbicide

HIV is a highly mutable virus that has become a global pandemic. In order to prevent further spread of this disease a topical microbicide that is cheap and easy to administer and produce is necessary. Our project involved the extraction of the HIV drug Nevirapine from commercial tablets to encapsulate it inside glucan particles for mucosal delivery. Glucan Particles (GP) are naturally internalized by macrophages and Langerhans cells thought to be the first cells to be HIV infected at mucosal sites. GP are hollow microparticles derived from baker\u27s yeast that can be used to absorb the Nevirapine into an encapsulated core. To keep the drug trapped inside the core various synthetic strategies were evaluated to identify an effective formulation for future in vitro and in vivo testing

Disain ja modelleerimine HIV-1 pöördtranskriptaasi ja Malaaria ravimite väljatöötamise varajases faasis

Väitekirja elektrooniline versioon ei sisalda publikatsiooneKäesolev uurimus keskendub kahele ohtlikule infektsioonhaigusele: inimese immuunpuudulikkuse viirus tüüp 1 (HIV-1) ja malaaria. Uue ravimi väljatöötamine algusest lõppuni on aega nõudev ning kulukas protsess, mis jaotatakse viieks etapiks: baas uurimistöö, põhi sihtmärgi ja baas ühendi(te) leidmine, eelkliiniline arendus, kliiniline arendus ja vajalike dokumentide esitamine ravimiametisse. Antud väitekirjas keskendutakse kahele esimesele etappidele, mida tuntakse ka varajase ravimiarenduse faasina. HIV-1 uurimisel oli kaks põhisuunda. Esmalt tuginedes eelnevalt tehtud virtuaalsõelumise tulemustele teostati uudsete s-triasiini derivaatide avastamine, disainimine, ja süntees, mille tulemused valideeriti eksperimentaalselt ning analüüsiti valk-ligand interaktsioonimudelite abil. Kõige tõhusam HIV-1 mitte-nukleosiidne pöördtranskriptaasi inhibiitor oli madala molekulmassiga, heade ligandi efektiivsust näitavate parameetritega, ja madala toksilisusega, võimaldades edasist modifitseerimist ja arendamist. Tehtud aktiivse keemilise struktuuri avastus motiveeris HIV-1 inhibiitorite keemilise struktuuriruumi laiemat uurimist, et kindlaks teha kas uudsed s-triasiinid moodustavad ka unikaalsed keemiliste ühendite grupi HIV-1 mitte-nukleosiidsete pöördtranskriptaasi inhibiitorite maastikul. Selle läbiviimiseks koostati, korrastati ja kureeriti ChEMBL-i andmebaasist saadud andmetest fokusseeritud andmeseeriad HIV-1 mitte-nukleosiidne ja nukelosiidsete pöördtranskriptaasi inhibiitorite jaoks, kuhu lisati ka avastatud s-triasiini derivaadid. Andmeseeriate struktuuride analüüs hierarhilise klassifitseerimise meetodil grupeeris ühendid keemiliste struktuuritüüpide (nn. vanematüüp) järgi. Selgus, et avastatud s-triasiinid moodustasid eraldiseisva struktuuritüübi grupi. Leitud struktuuritüüpe analüüsiti, lisades juurde ka vastavad mõõdetud seondumise afiinsuse tasakaalukonstandid (Ki). Selle analüüsi käigus toodi välja struktuurifragmendid, mis omavad olulist rolli afiinsuse ning stabiilsuse seisukohast. Lisaks võimaldasid struktuurselt mitmekesised ja unikaalsed HIV-1 mitte-nukleosiidne ja nukelosiidsete pöördtranskriptaasi inhibiitorite andmeseeriad esmakordselt arendada kirjeldavaid kvantitatiivsete struktuur-aktiivsus sõltuvuste prognoosmudeleid, mida on võimalik kasutada järgnevas uurimustöös uute aktiivsete keemiliste ühendite avastamisel. Selleks et leida uudseid malaaria ravimikanditaate koostati ja kureeriti süsteemselt andmebaas eksperimentaalsete anti-Plasmodium andmetega kasutades nii asutusesisesed, kui ka ChEMBL-i andmebaasis olevad andmed. Saadud andmete ulatusliku kureerimise, filtreerimise ning ühendamise tulemusena saadi kolmkümmend modelleeritavat andmeseeriat, millele koostati klassifitseerimise mudelid, eesmärgiga eristada aktiivsed ja mitteaktiivsed ühendid. Nendest seitsmeteistkümnele andmeseeriale saadi ennustusvõimelised nn. üksmeele (inglise keeles consensus) mudelid. Loodud mudelitega teostati ennustusi asutusesiseselt olemasolevatele curcuminoidide seerjale ning nende analoogidele, millest parima ennustusvõimega ühenditele teostati eksperimentaalne valideerimine in vitro katsetega, kus aktiivseks osutusid seitseteist ühendit, mida saab edasistes uuringutes täpsemini uurida. Samuti tehti kindaks, et arvutuslikult tuvastatud mitteaktiivsed ühendid jäid mitteaktiivseks ka eksperimentaalse valideerimise käigus, mis näitas süsteemselt kureeritud ja koostatud andmeseeriate ning prognoosmudelite jätkusuutlikust.Current thesis focused on study of two highly prevalent infections affecting many regions in the world: alaria and human immunodeficiency virus 1 (HIV-1). Developing a new drug from scratch is time consuming and costly process. This could be divided into five stages: basic research, lead target and lead compound(s) discovery, preclinical development, clinical development and filing to drug administration agency. Present thesis focused on basic research and lead compound discovery stages, i.e. to the early drug discovery. For the HIV-1, the focus was two-fold. First, based on the earlier multi-objective in silico screening, novel s-triazine derivatives were designed, discovered, synthesized, and findings where supported by the modelling tasks and validated with biological evaluation. The most potent compound is with small molecular size, potent ligand efficiencies, and measured low toxicity permitting further exploration and modifications. Second, the discovered new bioactive s-triazines motivated to analyse the chemical landscape of HIV-1 RT inhibitors. For this the dataset was systematically created and curated for HIV-1 NNRT (non-nucleoside reverse transcriptase) and NRT (nucleoside reverse transcriptase) inhibitors based on data from ChEMBL database. The hierarchical classification of scaffold structures of curated datasets revealed common chemical parent types for the compounds, hierarchy in chemical structures and showed that discovered s-triazines formed a separate structural parent type group. Each group of compounds related to the parent type was analysed and examined together with corresponding binding affinity equilibrium constants (Ki). The structural fragments affecting the potency and stability of compounds were highlighted. The structurally diverse datasets for the HIV-1 NNRTIs and NRTIs with binding affinity equilibrium constants allowed development of novel descriptive and predictive QSAR models for log Ki, that in future will help in design of new compounds. In order to discover new promising antimalarial compounds, the experimental anti-Plasmodium data was gathered and systematically curated from in-house experimental studies and expanded with data from ChEMBL database. Extracted data was carefully extensively curated, fused, filtered, and grouped into thirty data sets for the modelling. The consensus models for each dataset for the classification of active/inactive compounds were established and seventeen models with promising prediction ability were used in consensus predictions and in identifying the series of curcuminoids and their structural analogues as potential inhibitors for the malaria. The selection of compounds was experimentally validated, i.e. tested in vitro, revealing seventeen potentially active compounds for further testing and modifications. The validation showed that computationally predicted inactive compounds were also inactive in experiment, being additional proof for the quality of data curation and dataset assembly process forming the ground for the modelling task

Structure-based approaches applied to the study of pharmaceutical relevant targets

Computer Aided Drug Design/Discovery methods became complementary to traditional and modern drug discovery approaches. Indeed CADD is useful to improve and speed up the detection and the optimization of bioactive molecules. The present study is focused on the application of structure-based approaches to the study of pharmaceutical relevant targets. The introduction provides a quick overview on the fundamentals of computational chemistry and structure-based methods, while in the successive chapters the main targets investigated through these methods are treated. In particular we focused our attention on Reverse Transcriptase of HIV-1, Monoamine oxidase B and VP35 of Ebola virus. The last chapter is dedicated to the validation of covalent docking performed with Autodock