Paris Ward

Structure of the BTB Domain of Keap1 and Its Interaction with the Triterpenoid Antagonist CDDO

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis.

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.

Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors.

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

Aminoindazole PDK1 Inhibitors: A Case Study in Fragment-Based Drug Discovery.

Fragment screening of phosphoinositide-dependent kinase-1 (PDK1) in a biochemical kinase assay afforded hits that were characterized and prioritized based on ligand efficiency and binding interactions with PDK1 as determined by NMR. Subsequent crystallography and follow-up screening led to the discovery of aminoindazole 19, a potent leadlike PDK1 inhibitor with high ligand efficiency. Well-defined structure-activity relationships and protein crystallography provide a basis for further elaboration and optimization of 19 as a PDK1 inhibitor.

Acylprolinamides: a new class of peptide deformylase inhibitors with in vivo antibacterial activity.

A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.

Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.

Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85α–p110α fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors☆

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

Structure-guided optimization of small molecule c-Abl activators

Abstractc-Abl kinase is maintained in its normal inactive state in the cell through an assembled, compact conformation. We describe two chemical series that bind to the myristoyl site of the c-Abl kinase domain and stimulate c-Abl activation. We hypothesize that these molecules activate c-Abl either by blocking the C-terminal helix from adopting a bent conformation that is critical for the formation of the autoinhibited conformation or by simply providing no stabilizing interactions to the bent conformation of this helix. Structure-based molecular modeling guided the optimization of binding and activation of c-Abl of these two chemical series and led to the discovery of c-Abl activators with nanomolar potency. The small molecule c-Abl activators reported herein could be used as molecular tools to investigate the biological functions of c-Abl and therapeutic implications of its activation.