Andre White

Molecular basis of MAPK-activated protein kinase 2:p38 assembly

p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38α isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-Å resolution crystal structure of the unphosphorylated complex between p38α and MK2. These protein kinases bind “head-to-head,” present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366–Ala-390, which includes the bipartite nuclear localization signal, binds to the p38α-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38α binary assembly. The MK2 residues 345–365, containing the nuclear export signal, block access to the p38α active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38α and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein–protein interactions involving signal transduction proteins.

Novel and efficient chiral bisphosphorus ligands for rhodium-catalyzed asymmetric hydrogenation.

A series of structurally novel, operationally convenient, and efficient chiral 2-phosphino-2,3-dihydrobenzo[d][1,3]oxaphosphole ligands was developed. Applications of ligands 3a and 3b in rhodium-catalyzed asymmetric hydrogenation of alpha-(acylamino)acrylates and beta-(acylamino)acrylates provided excellent enantioselectivities (up to >99% ee) and reactivities (up to 10,000 TON).

Synthesis and SAR studies of indole-based MK2 inhibitors

Chemistry has been developed to specifically functionalize two structurally similar classes of indole-based MK2 inhibitors at positions prompted by a combination of X-ray crystallographic and computer assisted drug design. A gain in molecular potency was obtained by introducing aminomethyl groups to the lactam rings of 6-arylcarbamoyl-tetrahydro-beta-carbolinone and 6-arylcarbamoyl-dihydropyrazino[1,2-a]indolone MK2 inhibitors. In addition, improvements in molecular potency were achieved by expansion of the lactam from a 6- to 7-membered ring leading to 7-arylcarbamoyl-tetrahydro-[1,4]diazepino[1,2-a]indolones.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these ‘non-homeostatic’ conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the ‘emergency circuit’ that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

Discovery of potent inhibitors of interleukin-2 inducible T-cell kinase (ITK) through structure-based drug design

Interleukin-2 inducible T-cell kinase (ITK) is a member of the Tec kinase family and is involved with T-cell activation and proliferation. Due to its critical role in acting as a modulator of T-cells, ITK inhibitors could provide a novel route to anti-inflammatory therapy. This work describes the discovery of ITK inhibitors through structure-based design where high-resolution crystal structural information was used to optimize interactions within the kinase specificity pocket of the enzyme to improve both potency and selectivity.

Discovery, SAR and X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-amides as inhibitors of inducible T-cell kinase (Itk)

A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.

Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design.

Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low μM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).

RVX-297- a novel BD2 selective inhibitor of BET bromodomains.

Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. We demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains.

Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.