Complex peptide macrocycle optimization: combining NMR restraints with conformational analysis to guide structure-based and ligand-based design.

Systematic optimization of large macrocyclic peptide ligands is a serious challenge. Here, we describe an approach for lead-optimization using the PD-1/PD-L1 system as a retrospective example of moving from initial lead compound to clinical candidate. We show how conformational restraints can be derived by exploiting NMR data to identify low-energy solution ensembles of a lead compound. Such restraints can be used to focus conformational search for analogs in order to accurately predict bound ligand poses through molecular docking and thereby estimate ligand strain and protein-ligand intermolecular binding energy. We also describe an analogous ligand-based approach that employs molecular similarity optimization to predict bound poses. Both approaches are shown to be effective for prioritizing lead-compound analogs. Surprisingly, relatively small ligand modifications, which may have minimal effects on predicted bound pose or intermolecular interactions, often lead to large changes in estimated strain that have dominating effects on overall binding energy estimates. Effective macrocyclic conformational search is crucial, whether in the context of NMR-based restraints, X-ray ligand refinement, partial torsional restraint for docking/ligand-similarity calculations or agnostic search for nominal global minima. Lead optimization for peptidic macrocycles can be made more productive using a multi-disciplinary approach that combines biophysical data with practical and efficient computational methods.

Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors.

Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.

Conformation and absolute configuration of nocathiacin I determined by NMR spectroscopy and chiral capillary electrophoresis.

Nocathiacin I (BMS-249524) is a highly cross-linked thiazolyl peptide that displays potent activity against Gram-positive bacteria, including a number of antibiotic-resistant strains. This natural product contains 10 chiral centers. NMR studies have been performed to characterize the solution structure of nocathiacin I. A uniformly 13C,15N-labeled sample was used to obtain NMR assignments. Restrained simulated annealing calculations were performed by using accurately determined NOE distance restraints. All of the chiral centers were allowed to float during the simulated annealing protocol. Two clusters of structures were obtained that satisfy the NOE restraints very well and that are reasonably consistent with vicinal J-coupling constants. Within each cluster, all 10 chiral centers are uniquely defined. The two clusters are effectively mirror images of each other: all chiral centers that have the R(S) configuration in one cluster have the S(R) configuration in the other. The single threonine residue in nocathiacin I was subsequently determined to be l-threonine by chiral capillary electrophoresis, allowing the absolute configurations of all 10 chiral centers to be defined.