Structural characterization of a new N‐substituted pantothenamide bound to pantothenate kinases from Klebsiella pneumoniae and Staphylococcus aureus

Pantothenate kinase (PanK) is the rate‐limiting enzyme in Coenzyme A biosynthesis, catalyzing the ATP‐dependent phosphorylation of pantothenate. We solved the co‐crystal structures of PanKs from Staphylococcus aureus (SaPanK) and Klebsiella pneumonia (KpPanK) with N‐[2‐(1,3‐benzodioxol‐5‐yl)ethyl] pantothenamide (N354‐Pan). Two different N354‐Pan conformers interact with polar/nonpolar mixed residues in SaPanK and aromatic residues in KpPanK. Additionally, phosphorylated N354‐Pan is found at the closed active site of SaPanK but not at the open active site of KpPanK, suggesting an exchange of the phosphorylated product with a new N354‐Pan only in KpPanK. Together, pantothenamides conformational flexibility and binding pocket are two key considerations for selective compound design. Proteins 2014; 82:1542–1548. © 2014 Wiley Periodicals, Inc.     

A multiscale approach to predicting affinity changes in protein–protein interfaces

Substitution mutations in protein–protein interfaces can have a substantial effect on binding, which has consequences in basic and applied biomedical research. Experimental expression, purification, and affinity determination of protein complexes is an expensive and time‐consuming means of evaluating the effect of mutations, making a fast and accurate in silico method highly desirable. When the structure of the wild‐type complex is known, it is possible to economically evaluate the effect of point mutations with knowledge based potentials, which do not model backbone flexibility, but these have been validated only for single mutants. Substitution mutations tend to induce local conformational rearrangements only. Accordingly, ZEMu (Zone Equilibration of Mutants) flexibilizes only a small region around the site of mutation, then computes its dynamics under a physics‐based force field. We validate with 1254 experimental mutants (with 1–15 simultaneous substitutions) in a wide variety of different protein environments (65 protein complexes), and obtain a significant improvement in the accuracy of predicted ΔΔG. Proteins 2014; 82:2681–2690. © 2014 Wiley Periodicals, Inc.     

Physics‐based enzyme design: Predicting binding affinity and catalytic activity

Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical–chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics‐based implicit solvent MM‐GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM‐GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α‐Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real‐world enzyme design applications. Proteins 2014; 82:3397–3409. © 2014 Wiley Periodicals, Inc.     

Is there an associational resistance of winter pea–durum wheat intercrops towards Acyrthosiphon pisum Harris?

Acyrthosiphon pisum Harris (Aphididae: Hemiptera), the pea aphid, is an important pest in organic farming systems. In this work, the objective was to gather empirical field data on the associational resistance of durum wheat–winter pea intercrops towards the pea aphid, compared with pure stands of winter pea. Our results showed that intercropping winter pea with durum wheat significantly decreased A. pisum abundance in all the situations. Moreover, it was systematically observed that pea grew bigger in pure than in intercropped stands but after considering pea dry mass as a covariate, it appeared that the durum wheat–winter pea intercrop was still significantly less attacked by pea aphids than the sole crop. Intercrop sowing designs had an incidence on infestation levels: substitutive diversification systems of different types are more effective in decreasing the level of aphid infestation than does the additive system. In addition, substitutive row intercrop is significantly less infested than substitutive mixture. These results suggest that a mechanism related to the resource concentration hypothesis may explain the associational resistance of the IC of durum wheat–winter pea towards A. pisum.     

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective,orally available inhibitors of dipeptidyl peptidase IV

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.     

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.     

Structure-based design of novel human Pin1 inhibitors (III): Optimizing affinity beyond the phosphate recognition pocket

The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand–protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.     

Tetra-substituted imidazoles as a new class of inhibitors of the p53–MDM2 interaction

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53–MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein–protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.     

Green‐lighting green fluorescent protein: Faster and more efficient folding by eliminating a cis–trans peptide isomerization event

Wild‐type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis–trans peptide bond isomerization. The only conserved cis‐peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X‐P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans‐peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.