A novel chemogenomic discovery platform identifies bioactive hits with rapid bactericidal activity against Mycobacteroides Abscessus.

Mycobacteroides abscessus (M. ab) infections are innately resistant to most currently available antibiotics and present a growing, poorly addressed medical need. The existing treatment regimens are lengthy and produce inadequate outcomes for many patients. Importantly, most clinically used drugs and drug candidates against M. ab are either bacteriostatic, or only weakly bactericidal. New strategies exploring a broader chemical space are urgently needed, as innovative agents in development are scarce and hit rates in large unbiased screens against the mycobacterium have been discouragingly low. Here we present a computational chemogenomics-driven approach to discovery of novel antibacterials that effectively reveals drug-like compounds active against M. ab, paired with small sets of predicted molecular targets for the compounds. Several of the bioactive hits identified exhibited rapid bactericidal, including sterilizing, activity against the mycobacterium, indicating that there are currently unexploited chemically tractable molecular mechanisms for rapid sterilization of M. ab. Interestingly, starvation, which typically induces drug tolerance, sensitized M. ab to some of the compounds, resulting in potencies similar to those of drugs in clinical use. The presented drug discovery platform has potential to identify highly differentiated prototype anti-infective molecules and thereby contribute to development of regimens for shorter treatment and improved outcomes for non-tuberculous mycobacterial infections.

An integrated chemical biology approach provides insight into Cdk2 functional redundancy and inhibitor sensitivity.

Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition. Together with experiments in Xenopus egg extracts, this supports a rate-limiting role for Cdk2 in DNA replication. To confirm this we designed inhibitor-resistant (ir)-Cdk2 mutants using a novel bioinformatics approach. Bypassing inhibition with ir-Cdk2 or with Cdk1 shows that Cdk2 is rate-limiting for replication in this system because Cdk1 is insufficiently active. Additionally, crystal structures and kinetics reveal alternative binding modes of Cdk1-selective and Cdk2-selective inhibitors and mechanisms of Cdk2 inhibitor resistance. Our approach thus provides insight into structure, functions, and biochemistry of a cyclin-dependent kinase.

Prediction of the 3D structure and dynamics of human DP G-protein coupled receptor bound to an agonist and an antagonist.

Prostanoids play important physiological roles in the cardiovascular and immune systems and in pain sensation in peripheral systems through their interactions with eight G-protein coupled receptors. These receptors are important drug targets, but development of subtype specific agonists and antagonists has been hampered by the lack of 3D structures for these receptors. We report here the 3D structure for the human DP G-protein coupled receptor (GPCR) predicted by the MembStruk computational method. To validate this structure, we use the HierDock computational method to predict the binding mode for the endogenous agonist (PGD2) to DP. Based on our structure, we predicted the binding of different antagonists and optimized them. We find that PGD2 binds vertically to DP in the TM1237 region with the alpha chain toward the extracellular (EC) region and the omega chain toward the middle of the membrane. This structure explains the selectivity of the DP receptor and the residues involved in the predicted binding site correlate very well with available mutation experiments on DP, IP, TP, FP, and EP subtypes. We report molecular dynamics of DP in explicit lipid and water and find that the binding of the PGD2 agonist leads to correlated rotations of helices of TM3 and TM7, whereas binding of antagonist leads to no such rotations. Thus, these motions may be related to the mechanism of activation.

High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding.

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.

Sequence, structure and energetic determinants of phosphopeptide selectivity of SH2 domains.

Here, we present an approach for the prediction of binding preferences of members of a large protein family for which structural information for a number of family members bound to a substrate is available. The approach involves a number of steps. First, an accurate multiple alignment of sequences of all members of a protein family is constructed on the basis of a multiple structural superposition of family members with known structure. Second, the methods of continuum electrostatics are used to characterize the energetic contribution of each residue in a protein to the binding of its substrate. Residues that make a significant contribution are mapped onto the protein sequence and are used to define a "binding site signature" for the complex being considered. Third, sequences whose structures have not been determined are checked to see if they have binding-site signatures similar to one of the known complexes. Predictions of binding affinity to a given substrate are based on similarities in binding-site signature. An important component of the approach is the introduction of a context-specific substitution matrix suitable for comparison of binding-site residues. The methods are applied to the prediction of phosphopeptide selectivity of SH2 domains. To this end, the energetic roles of all protein residues in 17 different complexes of SH2 domains with their cognate targets are analyzed. The total number of residues that make significant contributions to binding is found to vary from nine to 19 in different complexes. These energetically important residues are found to contribute to binding through a variety of mechanisms, involving both electrostatic and hydrophobic interactions. Binding-site signatures are found to involve residues in different positions in SH2 sequences, some of them as far as 9A away from a bound peptide. Surprisingly, similarities in the signatures of different domains do not correlate with whole-domain sequence identities unless the latter is greater than 50%. An extensive comparison with the optimal binding motifs determined by peptide library experiments, as well as other experimental data indicate that the similarity in binding preferences of different SH2 domains can be deduced on the basis of their binding-site signatures. The analysis provides a rationale for the empirically derived classification of SH2 domains described by Songyang & Cantley, in that proteins in the same group are found to have similar residues at positions important for binding. Confident predictions of binding preference can be made for about 85% of SH2 domain sequences found in SWISSPROT. The approach described in this work is quite general and can, in principle, be used to analyze binding preferences of members of large protein families for which structural information for a number of family members is available. It also offers a strategy for predicting cross-reactivity of compounds designed to bind to a particular target, for example in structure-based drug design.

On the role of electrostatic interactions in the design of protein-protein interfaces.

Here, the methods of continuum electrostatics are used to investigate the contribution of electrostatic interactions to the binding of four protein-protein complexes; barnase-barstar, human growth hormone and its receptor, subtype N9 influenza virus neuraminidase and the NC41 antibody, the Ras binding domain (RBD) of kinase cRaf and a Ras homologue Rap1A. In two of the four complexes electrostatics are found to strongly oppose binding (hormone-receptor and neuraminidase-antibody complexes), in one case the net effect is close to zero (barnase-barstar) and in one case electrostatics provides a significant driving force favoring binding (RBD-Rap1A). In order to help understand the wide range of electrostatic contributions that were calculated, the electrostatic free energy was partitioned into contributions of individual charged and polar residues, salt bridges and networks involving salt bridges and hydrogen bonds. Although there is no one structural feature that accounts for the differences between the four interfaces, the extent to which the desolvation of buried charges is compensated by the formation of hydrogen bonds and ion pairs appears to be an important factor. Structural features that are correlated with contribution of an individual residue to stability are also discussed. These include partial burial of a charged group in the free monomer, the formation of networks involving charged and polar amino acids, and the formation of partially exposed ion-pairs. The total electrostatic contribution to binding is found to be inversely correlated with buried total and non-polar surface area. This suggests that different interfaces can be designed to exploit electrostatic and hydrophobic forces in very different ways.