Structural Determinants Of Selectivity Research Articles

Toward a Systematic Structural and Functional Annotation of Solute Carriers Transporters-Example of the SLC6 and SLC7 Families.

SLC transporters are emerging key drug targets. One important step for drug development is the profound understanding of the structural determinants defining the substrate selectivity of each transporter. Recently, the improvement of computational power and experimental methods such as X-ray and cryo-EM crystallography permitted to conduct structure-based studies on specific transporters having important pharmacological impact. However, a lot remains to be discovered regarding their dynamics, transport modulation and ligand recognition. A detailed functional characterization of transporters would provide opportunities to develop new compounds targeting these key drug targets. Here, we are giving an overview of two major human LeuT-fold families, SLC6 and SLC7, with an emphasis on the most relevant members of each family for drug development. We gather the most recent understanding on the structural determinants of selectivity within and across the two families. We then use this information to discuss the benefits of a more generalized structural and functional annotation of the LeuT fold and the implications of such mapping for drug discovery.

Profiling the Signaling Repertoire of One Hundred Therapeutically‐Relevant Human GPCRs

It is now recognized that a given GPCR can engage multiple signaling pathways and that specific ligands can selectively promote the engagement of different subsets of these pathways. To fully explore the potential of this functional selectivity phenomenon in drug discovery and development, it becomes essential to have an exhaustive description of the effectors that can be engaged by a given receptor, providing receptor‐ and ultimately ligand‐specific signaling signatures. Here, we describe a central resource for interrogating the signaling profiles of over 100 therapeutically relevant human GPCRs in response to their endogenous ligands. We used 15 pathway‐selective bioluminescence resonance energy transfer (BRET) biosensors monitoring the activation of each Gα protein, as well as βarrestins (−/+ co‐expressed GRK2). The G protein sensors used in the study represent a new generation of BRET‐based sensors that monitor the recruitment of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 concentration‐responses curves were generated and revealed a great diversity in the coupling selectivity of the studied receptors. Our data highlight that the Gi□family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Some GPCRs showed greater selectivity, with activation restricted to a single G protein subtype or members of the same G protein family (22% of tested GPCRs). Others showed pleiotropic activation profiles engaging G proteins belonging to multiple families (38% and 31% of tested receptors coupled to members of two and three G protein families, respectively) and, in some cases, all tested G proteins. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. This underappreciated level of receptor coupling selectivity raises fundamental questions on the structural determinants of selectivity among closely related G proteins. A similar receptor‐specific profile of activation was observed for barrestins with 74% of GPCRs recruiting βarrestin‐2, and co‐expression of GRK2 favoring this recruitment. This difference in functional selectivity for different GPCRs highlights the importance of testing the multiple pathways coupled to a receptor. Interestingly, our analysis reveals that monitoring the activation of Gz and G15 allowed to detect the activation for the vast majority of the 100 GPCRs tested. Consequently, combining Gz and G15 sensors provides a practical tool to detect the activity of almost any GPCR. Our BRET‐based biosensors also allow the detection of receptor constitutive activity, which can be applied for GPCR deorphanization or to identify new drugs targeting constitutively activated GPCRs overexpressed in cancer. Hence, in addition to describing and validating an exhaustive G protein activity sensor platform, the study provides an exhaustive description of the signaling repertoire of 100 therapeutically relevant GPCRs that may underlie their therapeutic efficacy/safety profiles.

The selectivity of galardin and an azasugar-based hydroxamate compound for human matrix metalloproteases and bacterial metalloproteases.

Inhibitors targeting bacterial enzymes should not interfere with enzymes of the host, and knowledge about structural determinants for selectivity is important for designing inhibitors with a therapeutic potential. We have determined the binding strengths of two hydroxamate compounds, galardin and compound 1b for the bacterial zinc metalloproteases, thermolysin, pseudolysin and auerolysin, known to be bacterial virulence factors, and the two human zinc metalloproteases MMP-9 and MMP-14. The active sites of the bacterial and human enzymes have huge similarities. In addition, we also studied the enzyme-inhibitor interactions by molecular modelling. The obtained Ki values of galardin for MMP-9 and MMP-14 and compound 1b for MMP-9 are approximately ten times lower than previously reported. Compound 1b binds stronger than galardin to both MMP-9 and MMP-14, and docking studies indicated that the diphenyl ether moiety of compound 1b obtains more favourable interactions within the S´1-subpocket than the 4-methylpentanoyl moiety of galardin. Both compounds bind stronger to MMP-9 than to MMP-14, which appears to be due to a larger S´1-subpocket in the former enzyme. Galardin, but not 1b, inhibits the bacterial enzymes, but the galardin Ki values were much larger than for the MMPs. The docking indicates that the S´1-subpockets of the bacterial proteases are too small to accommodate the diphenyl ether moiety of 1b, while the 4-methylpentanoyl moiety of galardin enters the pocket. The present study indicates that the size and shape of the ligand structural moiety entering the S´1-subpocket is an important determinant for selectivity between the studied MMPs and bacterial MPs.

Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

The α4βδ subtype of the γ-aminobutyric acid (GABA) type A receptors (GABAARs) has been shown to be implicated in high-affinity binding of the neuromodulator γ-hydroxybutyric acid (GHB), but may not be the only GHB high-affinity binding sites. Monastrol has been identified as a modulator of GHB high-affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined for modulation of GHB high-affinity binding using the GHB-specific ligand [3H]NCS-382 [(E,RS)-6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid] in rat brain homogenate. Only limited modifications were allowed on the monastrol scaffold in order to maintain modulation of GHB high-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [3H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high-affinity binding preferring modulator. However, compounds 34 and 37 were shown to be α1β2δ GABAAR selective modulators, without modulatory effects on GHB high-affinity binding. Thus, our study shows that minor modifications in the structure of monastrol affects the selectivity profile for the two targets under study enabling separation of the dual activity.

The Role of Thermodynamics in the Activity and Selectivity of Antimicrobial Peptides

Antimicrobial peptides (AMPs) are natural bactericidals and immunomodulators, and their action involves association to cell membranes and their perturbation. In vitro, they are selective for bacteria versus eukaryotic cells. Although AMPs are promising compounds to fight drug-resistant bacteria, many unanswered questions hinder their therapeutic application: is their main role bactericidal or immunomodulatory? What are the structural determinants of selectivity, other than their cationic charge? Is cell selectivity real or an artifact of the assay conditions?[1] By combining spectroscopic experiments on liposomes and cells and molecular dynamics simulations, we characterized how AMP activity and selectivity are regulated by multiple thermodynamic equilibria: membrane binding, aggregation, conformational transitions and orientation in the bilayer.[2] By measuring peptide association to bacteria and erythrocytes, we found that high coverage of the cell surface is needed to cause its death/lysis, showing that the carpet mechanism is relevant in real cells. Due to the binding equilibrium, the total peptide concentration in solution necessary to reach this threshold never decreases below micromolar values, even at low cell densities. These data indicate that immunomodulation might prevail where these concentrations are not reached, and provide a new insight in the cell density dependence of selectivity. In addition, we showed that peptide folding or aggregation in solution are ways to increase peptide selectivity, by shielding the hydrophobic residues and reducing the hydrophobic-driving force for binding to the neutral surface of eukaryotes. Overall, our studies indicate that characterization and modulation of the thermodynamic equilibria involved in peptide behavior are a valuable approach to understand AMP function and to improve their properties.1) NatChemBiol 2013 9:761; BiochimBiophysActa 2009 1788:1687.2) BiochimBiophysActa 2015 1848:581; ACS ChemBiol 2014 9:2003; JPeptSci. 2013 19:758; BiochimBiophysActa 2013 1828:1013; Biochemistry 2012 51:10124; CellMolLifeSci 2011 68:2281; BiophysJ 2010 99:1791.