Brian D. Hamman

A FRET-Based Assay Platform for Ultra-High Density Drug Screening of Protein Kinases and Phosphatases

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.

Novel Class of LIM-Kinase 2 Inhibitors for the Treatment of Ocular Hypertension and Associated Glaucoma

The discovery of a pyrrolopyrimidine class of LIM-kinase 2 (LIMK2) inhibitors is reported. These LIMK2 inhibitors show good potency in enzymatic and cellular assays and good selectivity against ROCK. After topical dosing to the eye in a steroid induced mouse model of ocular hypertension, the compounds reduce intraocular pressure to baseline levels. The compounds also increase outflow facility in a pig eye perfusion assay. These results suggest LIMK2 may be an effective target for treating ocular hypertension and associated glaucoma.

Profound Obesity Secondary to Hyperphagia in Mice Lacking Kinase Suppressor of Ras 2

The kinase suppressor of ras 2 (KSR2) gene resides at human chromosome 12q24, a region linked to obesity and type 2 diabetes (T2D). While knocking out and phenotypically screening mouse orthologs of thousands of druggable human genes, we found KSR2 knockout (KSR2−/−) mice to be more obese and glucose intolerant than melanocortin 4 receptor−/− (MC4R−/−) mice. The obesity and T2D of KSR2−/− mice resulted from hyperphagia which was unresponsive to leptin and did not originate downstream of MC4R. The kinases AMP‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are each linked to food intake regulation, but only mTOR had increased activity in KSR2−/− mouse brain, and the ability of rapamycin to inhibit food intake in KSR2−/− mice further implicated mTOR in this process. The metabolic phenotype of KSR2 heterozygous (KSR2+/minus;) and KSR2−/− mice suggests that human KSR2 variants may contribute to a similar phenotype linked to human chromosome 12q24.

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

Binding of a Pleckstrin Homology Domain Protein to Phosphoinositide in Membranes: A Miniaturized FRET-Based Assay for Drug Screening

Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton’s tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP3) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP3, with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP3 at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP3 and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP3 in vesicles. This biochemical assay readily miniaturized to 1.8-μl reaction volumes and was validated in a 3456-well screening format.

A Homogeneous G Protein-Coupled Receptor Ligand Binding Assay Based on Time-Resolved Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) has emerged as a powerful tool to the study of protein-protein interactions, such as receptor-ligand binding. However, the application of FRET to the study of G protein-coupled receptors (GPCRs) has been limited by the method of labeling receptor with fluorescence probes. Here we described a novel time-resolved (TR)-FRET method to study GPCR-ligand binding by using human complement 5a (C5a) receptor (C5aR) as a model system. Human C5aR was expressed in human embryonic kidney 293 cells with a hemagglutinin (HA) epitope at the N-terminus. Purified human C5a was labeled with terbium chelate and used as the fluorescence donor. Monoclonal anti-HA antibody conjugated with Alexa Fluor 488 was used as the fluorescence acceptor. Robust FRET signal was observed when the labeled ligand and C5aR membrane were mixed in the presence of the conjugated anti-HA antibody. This FRET signal was specific and saturable. C5a binding affinity to C5aR measured by the FRET assay was consistent with the data as determined by competition binding analysis using radiolabeled C5a. The FRET assay was also used to determine affinity of C5aR antagonists by competition binding analysis, and the data are similar to those from radioligand binding studies. Compared to the commonly used radioligand binding assay, this TR-FRET-based assay provides a nonradioactive, faster, and sensitive homogeneous assay format that could be easily adapted to high-throughput screening. The principle of this assay should also be applicable to other GPCRs, especially to those receptors with peptide or protein ligands.

Discovery and Development of LX7101, a Dual LIM-Kinase and ROCK Inhibitor for the Treatment of Glaucoma.

The structure of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of ocular hypertension and associated glaucoma, is disclosed. Previously reported LIM kinase inhibitors suffered from poor aqueous stability due to solvolysis of the central urea. Replacement of the urea with a hindered amide resulted in aqueous stable compounds, and addition of solubilizing groups resulted in a set of compounds with good properties for topical dosing in the eye and good efficacy in a mouse model of ocular hypertension. LX7101 was selected as a clinical candidate from this group based on superior efficacy in lowering intraocular pressure and a good safety profile. LX7101 completed IND enabling studies and was tested in a Phase 1 clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure.

Discovery of a Type III Inhibitor of LIM Kinase 2 That Binds in a DFG-Out Conformation.

The first allosteric, type III inhibitor of LIM-kinase 2 (LIMK2) is reported. A series of molecules that feature both an N-phenylsulfonamide and tertiary amide were not only very potent at LIMK2 but also were extremely selective against a panel of other kinases. Enzymatic kinetic studies showed these molecules to be noncompetitive with ATP, suggesting allosteric inhibition. X-ray crystallography confirmed that these sulfonamides are a rare example of a type III kinase inhibitor that binds away from the highly conserved hinge region and instead resides in the hydrophobic pocket formed in the DFG-out conformation of the kinase, thus accounting for the high level of selectivity observed.

Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.

A High-Throughput Screen for Receptor Protein Tyrosine Phosphatase–γ Selective Inhibitors

Protein tyrosine phosphatase–γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z′-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC50 determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC50 value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.