Kinetic and dynamical properties of truncated hemoglobins of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Assessment of Embedding Schemes in a Hybrid Machine Learning/Classical Potentials (ML/MM) Approach.

Machine learning (ML) methods have reached high accuracy levels for the prediction of in vacuo molecular properties. However, the simulation of large systems solely through ML methods (such as those based on neural network potentials) is still a challenge. In this context, one of the most promising frameworks for integrating ML schemes in the simulation of complex molecular systems are the so-called ML/MM methods. These multiscale approaches combine ML methods with classical force fields (MM), in the same spirit as the successful hybrid quantum mechanics-molecular mechanics methods (QM/MM). The key issue for such ML/MM methods is an adequate description of the coupling between the region of the system described by ML and the region described at the MM level. In the context of QM/MM schemes, the main ingredient of the interaction is electrostatic, and the state of the art is the so-called electrostatic-embedding. In this study, we analyze the quality of simpler mechanical embedding-based approaches, specifically focusing on their application within a ML/MM framework utilizing atomic partial charges derived in vacuo. Taking as reference electrostatic embedding calculations performed at a QM(DFT)/MM level, we explore different atomic charges schemes, as well as a polarization correction computed using atomic polarizabilites. Our benchmark data set comprises a set of about 80k small organic structures from the ANI-1x and ANI-2x databases, solvated in water. The results suggest that the minimal basis iterative stockholder (MBIS) atomic charges yield the best agreement with the reference coupling energy. Remarkable enhancements are achieved by including a simple polarization correction.

Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

Catalytic Mechanism of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A.

The oxidation of Met to methionine sulfoxide (MetSO) by oxidants such as hydrogen peroxide, hypochlorite, or peroxynitrite has profound effects on protein function. This modification can be reversed by methionine sulfoxide reductases (msr). In the context of pathogen infection, the reduction of oxidized proteins gains significance due to microbial oxidative damage generated by the immune system. For example, Mycobacterium tuberculosis (Mt) utilizes msrs (MtmsrA and MtmsrB) as part of the repair response to the host-induced oxidative stress. The absence of these enzymes makes Mycobacteria prone to increased susceptibility to cell death, pointing them out as potential therapeutic targets. This study provides a detailed characterization of the catalytic mechanism of MtmsrA using a comprehensive approach, including experimental techniques and theoretical methodologies. Confirming a ping-pong type enzymatic mechanism, we elucidate the catalytic parameters for sulfoxide and thioredoxin substrates (kcat/KM = 2656 ± 525 M-1 s-1 and 1.7 ± 0.8 × 106 M-1 s-1, respectively). Notably, the entropic nature of the activation process thermodynamics, representing ∼85% of the activation free energy at room temperature, is underscored. Furthermore, the current study questions the plausibility of a sulfurane intermediate, which may be a transition-state-like structure, suggesting the involvement of a conserved histidine residue as an acid-base catalyst in the MetSO reduction mechanism. This mechanistic insight not only advances our understanding of Mt antioxidant enzymes but also holds implications for future drug discovery and biotechnological applications.

The carbonyl-lock mechanism underlying non-aromatic fluorescence in biological matter.

Challenging the basis of our chemical intuition, recent experimental evidence reveals the presence of a new type of intrinsic fluorescence in biomolecules that exists even in the absence of aromatic or electronically conjugated chemical compounds. The origin of this phenomenon has remained elusive so far. In the present study, we identify a mechanism underlying this new type of fluorescence in different biological aggregates. By employing non-adiabatic ab initio molecular dynamics simulations combined with a data-driven approach, we characterize the typical ultrafast non-radiative relaxation pathways active in non-fluorescent peptides. We show that the key vibrational mode for the non-radiative decay towards the ground state is the carbonyl elongation. Non-aromatic fluorescence appears to emerge from blocking this mode with strong local interactions such as hydrogen bonds. While we cannot rule out the existence of alternative non-aromatic fluorescence mechanisms in other systems, we demonstrate that this carbonyl-lock mechanism for trapping the excited state leads to the fluorescence yield increase observed experimentally, and set the stage for design principles to realize novel non-invasive biocompatible probes with applications in bioimaging, sensing, and biophotonics.

Minimum Free Energy Pathways of Reactive Processes with Nudged Elastic Bands.

The determination of minimum free energy pathways (MFEP) is one of the most widely used strategies to study reactive processes. For chemical reactions in complex environments, the combination of quantum mechanics (QM) with a molecular mechanics (MM) representation is usually necessary in a hybrid QM/MM framework. However, even within the QM/MM approximation, the affordable sampling of the phase space is, in general, quite restricted. To reduce drastically the computational cost of the simulations, several methods such as umbrella sampling require performing a priori a selection of a reaction coordinate. The quality of the computed results, in an affordable computational time, is intimately related to the reaction coordinate election which is, in general, a nontrivial task. In this work, we provide an approach to model reactive processes in complex environments that does not require the a priori selection of a reaction coordinate. The proposed methodology combines QM/MM simulations with an extrapolation of the nudged elastic bands (NEB) method to the free energy surface (FENEB). We present and apply our own FENEB scheme to optimize MFEP in different reactive processes, using QM/MM frameworks at semiempirical and density functional theory levels. Our implementation is based on performing the FENEB optimization by uncoupling the optimization of the band in a perpendicular and tangential direction. In each step, a full optimization with the spring force is performed, which guarantees that the images remain evenly distributed. The robustness of the method and the influence of sampling on the quality of the optimized MFEP and its associated free energy barrier are studied. We show that the FENEB method provides a good estimation of the reaction barrier even with relatively short simulation times, supporting that its combination with QM/MM frameworks provides an adequate tool to study chemical processes in complex environments.

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

Binding mechanism of disulfide species to ferric hemeproteins: The case of metmyoglobin.

The interactions of the heme iron of hemeproteins with sulfide and disulfide compounds are of potential interest as physiological signaling processes. While the interaction with hydrogen sulfide has been described computationally and experimentally, the reaction with disulfide, and specifically the molecular mechanism for ligand binding has not been studied in detail. In this work, we study the association process for disulfane and its conjugate base disulfanide at different pH conditions. Additionally, by means of advanced sampling techniques based on multiple steered molecular dynamics, we provide free energy profiles for ligand migration for both acid/base species, showing a similar behavior to the previously reported for the related H2S/HS¯ pair. Finally, we studied the ligand interchange reaction (H2O/H2S, HS¯ and H2O/HSSH, HSS¯) by means of hybrid quantum mechanics-molecular mechanics calculations. We show that the anionic species are able to displace more efficiently the H2O bound to the iron, and that the H-bond network in the distal cavity can help the neutral species to perform the reaction. Altogether, we provide a molecular explanation for the experimental information and show that the global association process depends on a fine balance between the migration towards the active site and the ligand interchange reaction.

Reduction of metmyoglobin by inorganic disulfide species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Novel Lennard-Jones Parameters for Cysteine and Selenocysteine in the AMBER Force Field.

Cysteine is a common amino acid with a thiol group that plays a pivotal role in a variety of scenarios in redox biochemistry. In contrast, selenocysteine, the 21st amino acid, is only present in 25 human proteins. Classical force-field parameters for cysteine and selenocysteine are still scarce. In this context, we present a methodology to obtain Lennard-Jones parameters for cysteine and selenocysteine in different physiologically relevant oxidation and protonation states. The new force field parameters obtained in this work are available at https://github.com/MALBECC/AMBER-parameters-database. The parameters were adjusted to reproduce water radial distribution functions obtained by density functional theory ab initio molecular dynamics. We validated the results by evaluating the impact of the choice of parameters on the structure and dynamics in classical molecular dynamics simulations of representative proteins containing catalytic cysteine/selenocysteine residues. There are significant changes in protein structure and dynamics depending on the parameters choice, specifically affecting the residues close to the catalytic sites.

Possible molecular basis of the biochemical effects of cysteine-derived persulfides.

Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.

Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study.

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Reactivity of inorganic sulfide species towards a pentacoordinated heme model system.

The reactivity of inorganic sulfide towards ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate has been explored by means of visible absorption and resonance Raman spectroscopies. The reaction has been previously studied in buffered solutions at neutral pH and in the presence of excess sulfide, revealing the formation of a moderately stable hexacoordinated low spin ferric sulfide complex that yields the ferrous form in the hour's timescale. In the surfactant solution, instead, the ferrous form is rapidly formed. The spectroscopic characterization of the heme structure in the surfactant milieu revealed the stabilization of a major ferric mono-histidyl high spin heme, which may be ascribed to out of plane distortions prompting the detachment of the axially ligated water molecule, thus leading to a differential reactivity. The ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate provides a model for pentacoordinated heme platforms with an imidazole-based ligand.

Acidity and nucleophilic reactivity of glutathione persulfide.

Persulfides (RSSH/RSS-) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS-), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS- presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, ßnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.

Nucleotide-dependent dynamics of the Dengue NS3 helicase.

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.

In Silico Insight into the Reductive Nitrosylation of Ferric Hemeproteins.

A combination of in silico methods was used to extend the experimental description of the reductive nitrosylation mechanism in ferric hemeproteins with the molecular details of the role of surrounding amino acids. The computational strategy consisted in the estimation of potential energy profiles for the transition process associated with the interactions of the coordinated N(NO) moiety with O(H2O) or O(OH-) as nucleophiles, and with distal amino acids as proton acceptors or affecting the stability of transition states. We inspected the reductive nitrosylation in three representative hemeproteins -sperm whale metmyoglobin, α subunit of human methemoglobin and nitrophorin 4 of Rhodnius prolixus. For each case, classical molecular dynamics simulations were performed in order to obtain relevant reactive conformations, and a potential energy profile for the reactive step was obtained using adiabatic mapping or nudged elastic band approaches at the QM/MM level. Specifically, we report the role of a charged Arg45 of myoglobin in destabilizing the transition state when H2O acts as nucleophile, differently to the neutral Pro43 of the hemoglobin subunit. The case of the nitrophorin is unique in that the access of the required water molecules is scarce, thus, preventing the reaction.

Dioxygen Binding and Sensing Proteins.

Oxygen binding proteins (O2BIP) have been actively investigated for the past five decades due to their rich redox chemistry and function as O2 carriers in blood cells, as well as their function as gasotransmitters and sensors that modulate cellular signaling. A series of meetings on the periodic advances in the knowledge gained in the field of globin structure and function are conducted typically on a biannual basis. In the fall of 2018, the XXth International Conference was conducted, and very important articles with breakthrough discoveries were presented and very enthusiastically discussed. This was yet another highly successful meeting in the series. Select articles from this meeting were recently reviewed, updated, and published over several issues of Antioxidants and Redox Signaling, as Forum articles communicating the latest advances in this important area of redox biology. This Forum editorial introduces these articles and highlights their scientific significance in advancing the field. Each of these articles grew out of lectures presented in the meeting, and appears either as an original contribution or a comprehensive review in the journal. Overall, the articles published in the Forum provide in-depth details on the recent developments in the field as well as point the way to future directions. These Forum articles thus serve as an important summary of progress and the ongoing direction of this field, and serve to highlight recent advances in our understanding of O2BIP.

3-Nitrotyrosine and related derivatives in proteins: precursors, radical intermediates and impact in function.

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical-radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO-), the product of the reaction of superoxide radical (O2•-) with •NO, but ONOO--independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.

Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents targeting reverse transcriptase enzyme.

Infection by human immunodeficiency virus still represents a continuous serious concern and a global threat to human health. Due to appearance of multi-resistant virus strains and the serious adverse side effects of the antiretroviral therapy administered, there is an urgent need for the development of new treatment agents, more active, less toxic and with increased tolerability to mutations. Quinoxaline derivatives are an emergent class of heterocyclic compounds with a wide spectrum of biological activities and therapeutic applications. These types of compounds have also shown high potency in the inhibition of HIV reverse transcriptase and HIV replication in cell culture. For these reasons we propose, in this work, the design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV reverse transcriptase enzyme. For this, we first carried out a structure-based development of target-specific compound virtual chemical library of quinoxaline derivatives. The rational construction of the virtual chemical library was based on previously assigned pharmacophore features. This library was processed by a virtual screening protocol employing molecular docking and 3D-QSAR. Twenty-five quinoxaline compounds were selected for synthesis in the basis of their docking and 3D-QSAR scores and chemical synthetic simplicity. They were evaluated as inhibitors of the recombinant wild-type reverse transcriptase enzyme. Finally, the anti-HIV activity and cytotoxicity of the synthesized quinoxaline compounds with highest reverse transcriptase inhibitory capabilities was evaluated. This simple screening strategy led to the discovery of two selective and potent quinoxaline reverse transcriptase inhibitors with high selectivity index.