Charles Belunis

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules

We have identified a heptapeptide with high affinity to rheumatoid arthritis–associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Deconstruction of a Nutlin: Dissecting the Binding Determinants of a Potent Protein–Protein Interaction Inhibitor

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.

Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: insights into rational drug design and activity regulation.

IL‐2‐inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2‐mediated inflammatory diseases. By applying high‐throughput protein engineering and crystallization, we have determined the X‐ray crystal structures of IL‐2‐inducible T cell kinase in complex with its selective inhibitor BMS‐509744 and the broad‐spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL‐2‐inducible T cell kinase in the helix C‐in conformation by inducing side chain conformational changes in the ATP‐binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad‐spectrum kinase activity. BMS‐509744 uniquely stabilizes the activation loop in a substrate‐blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL‐2‐inducible T cell kinase activity. The observed BMS‐509744 binding mode allows rationalization of structure–activity relationships reported for this inhibitor class and facilitates further structure‐based drug design. Sequence‐based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.

Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.

The structure of XIAP BIR2: understanding the selectivity of the BIR domains.

The high-resolution crystal structures of apo and peptide-bound XIAP BIR2 are presented and compared with BIR3 structures to understand their selectivity. This crystal system can be used to determine the structures of BIR2–inhibitor complexes.