Karina Martínez-Mayorga

Recognizing Pitfalls in Virtual Screening: A Critical Review

The aim of virtual screening (VS) is to identify bioactive compounds through computational means, by employing knowledge about the protein target (structure-based VS) or known bioactive ligands (ligand-based VS). In VS, a large number of molecules are ranked according to their likelihood to be bioactive compounds, with the aim to enrich the top fraction of the resulting list (which can be tested in bioassays afterward). At its core, VS attempts to improve the odds of identifying bioactive molecules by maximizing the true positive rate, that is, by ranking the truly active molecules as high as possible (and, correspondingly, the truly inactive ones as low as possible). In choosing the right approach, the researcher is faced with many questions: where does the optimal balance between efficiency and accuracy lie when evaluating a particular algorithm; do some methods perform better than others and in what particular situations; and what do retrospective results tell us about the prospective utility of a particular method? Given the multitude of settings, parameters, and data sets the practitioner can choose from, there are many pitfalls that lurk along the way which might render VS less efficient or downright useless. This review attempts to catalogue published and unpublished problems, shortcomings, failures, and technical traps of VS methods with the aim to avoid pitfalls by making the user aware of them in the first place.

Two protonation switches control rhodopsin activation in membranes

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV–visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to ≈50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.

Visualization of the Chemical Space in Drug Discovery

Chemical space has become a key concept in drug discovery. The continued growth in the number of molecules available raises the question regarding how many compounds may exist and which ones have the potential to become drugs. Analysis and visualization of the chemical space covered by public, commercial, in-house and virtual compound collections have found multiple applications in diversity analysis, in silico property profiling, data mining, virtual screening, library design, prioritization in screening campaigns, and acquisition of compound collections, among others. This review covers several techniques, computational programs and approaches that have been developed to visualize, navigate and study the chemical space of molecular databases. Techniques developed in our group are presented including a quantitative assessment of the multi-fusion similarity maps. Additionally an application of 3D-similarity, based on the overlay of chemical structures, to represent the chemical space is introduced. Several comparisons of the chemical space covered by compound collections from different sources such as combinatorial libraries, drugs and natural products, or directed to specific therapeutic areas are also discussed.

Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation

X-ray and magnetic resonance approaches, though central to studies of G protein–coupled receptor (GPCR)-mediated signaling, cannot address GPCR protein dynamics or plasticity. Here we show that solid-state 2H NMR relaxation elucidates picosecond-to-nanosecond–timescale motions of the retinal ligand that influence larger-scale functional dynamics of rhodopsin in membranes. We propose a multiscale activation mechanism whereby retinal initiates collective helix fluctuations in the meta I–meta II equilibrium on the microsecond-to-millisecond timescale.

Multitarget Structure–Activity Relationships Characterized by Activity-Difference Maps and Consensus Similarity Measure

Dual and triple activity-difference (DAD/TAD) maps are tools for the systematic characterization of structure-activity relationships (SAR) of compound data sets screened against two or three targets. DAD and TAD maps are two- and three- dimensional representations of the pairwise activity differences of compound data sets, respectively. Adding pairwise structural similarity information into these maps readily reveals activity cliff regions in the SAR for one, two, or three targets. In addition, pairs of compounds in the smooth regions of the SAR and scaffold hops are also easily identified in these maps. Herein, DAD and TAD maps are employed for the systematic characterization of the SAR of a benchmark set of 299 compounds screened against dopamine, norepinephrine, and serotonin transporters. To reduce the well-known dependence of the activity landscape on the structural representation, five selected 2D and 3D structure representations were used to characterize the SAR. Systematic analysis of the DAD and TAD maps reveals regions in the landscape with similar SAR for two or the three targets as well as regions with inverse SAR, i.e., changes in structure that increase activity for one target, but decrease activity for the other target. Focusing the analysis on pairs of compounds with high structure similarity revealed the presence of single-, dual-, and triple-target activity cliffs, i.e., small changes in structure with high changes in potency for one, two, or the three targets, respectively. Triple-target scaffold hops are also discussed. Activity cliffs and scaffold hops were also quantified and represented using two recently proposed approaches namely, mean Structure Activity Landscape Index (mean SALI) and Consensus Structure-Activity Similarity (SAS) maps.

Integrating Virtual Screening and Combinatorial Chemistry for Accelerated Drug Discovery

Virtual screening is increasingly being used in drug discovery programs with a growing number of successful applications. Experimental methodologies developed to speed up the drug discovery processes include high-throughput screening and combinatorial chemistry. The complementarities between computational and experimental screenings have been recognized and reviewed in the literature. Computational methods have also been used in the combinatorial chemistry field, in particular in library design. However, the integration of computational and combinatorial chemistry screenings has been attempted only recently. Combinatorial libraries (experimental or virtual) represent a notable source of chemically related compounds. Advances in combinatorial chemistry and deconvolution strategies, have enabled the rapid exploration of novel and dense regions in the chemical space. The present review is focused on the integration of virtual and experimental screening of combinatorial libraries. Applications of virtual screening to discover novel anticancer agents and our ongoing efforts towards the integration of virtual screening and combinatorial chemistry are also discussed.

Synthesis, in vitro and computational studies of protein tyrosine phosphatase 1B inhibition of a small library of 2-arylsulfonylaminobenzothiazoles with antihyperglycemic activity

The 2-arylsulfonylaminobenzothiazole derivatives 1-27 were prepared using a one step reaction. The in vitro inhibitory activity of the compounds against protein tyrosine phosphatase 1B (PTP-1B) was evaluated. Compounds 4 and 16 are rapid reversible (mixed-type) inhibitors of PTP-1B with IC(50) values in the low micromolar range. The most active compounds (4 and 16) were docked into the crystal structure of PTP-1B. Docking results indicate potential hydrogen bond interactions between the nitro group in both compounds and the catalytic amino acid residues Arg 221 and Ser 216. Both compounds were evaluated for their in vivo antihyperglycemic activity in a type 2 diabetes mellitus rat model, showing significant lowering of plasma glucose concentration, during the 7h post-intragastric administration.

Chemoinformatic analysis of GRAS (Generally Recognized as Safe) flavor chemicals and natural products.

Food materials designated as “Generally Recognized as Safe” (GRAS) are attracting the attention of researchers in their attempts to systematically identify compounds with putative health-related benefits. In particular, there is currently a great deal of interest in exploring possible secondary benefits of flavor ingredients, such as those relating to health and wellness. One step in this direction is the comprehensive characterization of the chemical structures contained in databases of flavoring substances. Herein, we report a comprehensive analysis of the recently updated FEMA GRAS list of flavoring substances (discrete chemical entities only). Databases of natural products, approved drugs and a large set of commercial molecules were used as references. Remarkably, natural products continue to be an important source of bioactive compounds for drug discovery and nutraceutical purposes. The comparison of five collections of compounds of interest was performed using molecular properties, rings, atom counts and structural fingerprints. It was found that the molecular size of the GRAS flavoring substances is, in general, smaller cf. members of the other databases analyzed. The lipophilicity profile of the GRAS database, a key property to predict human bioavailability, is similar to approved drugs. Several GRAS chemicals overlap to a broad region of the property space occupied by drugs. The GRAS list analyzed in this work has high structural diversity, comparable to approved drugs, natural products and libraries of screening compounds. This study represents one step towards the use of the distinctive features of the flavoring chemicals contained in the GRAS list and natural products to systematically search for compounds with potential health-related benefits.

Pyridin-2(1H)-ones: A Promising Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors

The acquired immune deficiency syndrome (AIDS) continues to be a major health problem worldwide with approximately 40 million people infected with the human immunodeficiency virus (HIV). A vast amount of research has lead to the identification of several molecular targets for the treatment of HIV infections. Current approved drugs are targeted to the retroviral enzymes reverse transcriptase (RT) and protease, and there is one fusion inhibitor. Treatment of AIDS involves a combination of three or more drugs in a regimen called highly active antiretroviral treatment (HAART), typically including RT and protease inhibitors. Despite the fact that HAART has significantly improved the levels of patient mortality, there are several problems to solve, with the emergence of clinical resistance a major challenge. The enzyme RT is responsible for the conversion of singlestranded RNA viral genome into a double-stranded DNA copy. RT inhibitors are classified as nucleoside and non-nucleoside, depending on their mechanism of action. Nucleoside inhibitors bind and inhibit the active site through chain termination of DNA synthesis whereas the non-nucleoside binds to a hydrophobic nonactive site pocket, about 10 ; away from the catalytic site (Figure 1). Non-nucleoside RT inhibitors (NNRTIs) are very attractive as anti-HIV agents because of their high selectivity, relatively low toxicity, and activity in the nanomolar range. Three NNRTIs have been approved for clinical use namely, nevirapine (VIRAMUNE), delavirdine (RESCRIPTOR), and efavirenz (SUSTIVA, STOCRIN) approved in 1996, 1997, and 1998, respectively (Figure 2). However, all these induce drug resistant variants of HIV-1. Examples of common mutations in the binding site of NNRTIs that confer resistance are Y181C, Y188C, K103N, and L100 A. Significant progress has been made in the search for the next generation of NNRTIs able to overcome resistance mutations, which are a major challenge in the design of new NNRTIs. Examples of such efforts is the development of TMC-125 (etravirine) (Figure 2) and the structurally related TMC-278 (rilpivirine) that are currently in phase III and phase II clinical trials, respectively. 5] A second example is the potent RT inhibitor UC781 (Figure 2) active against several mutant strains. This molecule is being developed as a microbicide to prevent HIV-1 transmission. In addition to approved drugs and compounds in clinical studies, there are several other molecules, covering more than 50 different structural classes, that have been identified as NNRTIs. The reader is referred to recent excellent reviews regarding NNRTIs in Ref. [4– 7] and references therein. Pyridin-2(1H)-ones, a class of NNRTIs, were discovered in a screening program at Merck and the first potent analogues advanced into clinical trials. Despite the development of Merck pyridinones being suspended because of the emergence of resistance, the pyridin-2(1H)-one ring remained a promising core Figure 1. Structure of HIV-1 reverse transcriptase. Residues of the active site are represented as a light gray surface and residues of the NNRTIs binding site as a dark gray surface.

Retinal Ligand Mobility Explains Internal Hydration and Reconciles Active Rhodopsin Structures

Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state (2)H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp265(6.48) residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.