Glenn A. Hofmann

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

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.

Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Two new cytotoxic cytochalasins from Xylaria obovata

Abstract Two new cytotoxic cytochalasins were isolated by brine shrimp bioassay-guided fractionation from a culture of the wood inhabiting fungus, Xylaria obovata . Their structures were determined as 19,20-epoxycytochalasin Q ( 1 ) and its deacetyl analog 2 by the application of spectroscopic techniques and chemical correlation with cytochalasin R. Acetylation of 2 yielded 1 . Both 1 and 2 were cytotoxic but were found to be inactive in an HIV-protease inhibitory assay and a mechanism-based DNA damaging yeast assay.

Process Validation and Screen Reproducibility in High-Throughput Screening

The use of large-scale compound screening has become a key component of drug discovery projects in both the pharmaceutical and the biotechnological industries. More recently, these activities have also been embraced by the academic community as a major tool for chemical genomic activities. High-throughput screening (HTS) activities constitute a major step in the initial drug discovery efforts and involve the use of large quantities of biological reagents, hundreds of thousands to millions of compounds, and the utilization of expensive equipment. All these factors make it very important to evaluate in advance of the HTS campaign any potential issues related to reproducibility of the experimentation and the quality of the results obtained at the end of these very costly activities. In this article, the authors describe how GlaxoSmithKline (GSK) has addressed the need of a true validation of the HTS process before embarking in full HTS campaigns. They present 2 different aspects of the so-called validation process: (1) optimization of the HTS workflow and its validation as a quality process and (2) the statistical evaluation of the HTS, focusing on the reproducibility of results and the ability to distinguish active from nonactive compounds in a vast collection of samples. The authors describe a variety of reproducibility indexes that are either innovative or have been adapted from generic medical diagnostic screening strategies. In addition, they exemplify how these validation tools have been implemented in a number of case studies at GSK. (Journal of Biomolecular Screening 2009:66-76)

Z-Axinohydantoin and Debromo-Z-Axinohydantoin from the Sponge Stylotella aurantium: Inhibitors of Protein Kinase C

Abstract During the large scale isolation of the hymenialdisine and debromohymenialdisine, potent inhibitors of PKC, from Stylotella aurantium, we also isolated several related minor metabolites, two of which proved to be novel. The structures of these new compounds, Z-axinohydantoin (1) and debromo-Z-axinohydantoin (2), were determined by interpretation of spectral data. Z-Axinohydantoin (1) and debromo-Z-axinohydantoin (2), were found to be inhibitors of PKC with IC50values of 9.0 and 22 μM respectively.

Topoisomerase II in multiple drug resistance

Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5′) or noncovalently (3′) to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types, i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.

Assay Development and High-Throughput Screening of Small Molecular c-Abl Kinase Activators

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC50 <10 µM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB2) receptor. The results were consistent with changes in cAMP content of hCB2-transfected human embryonic kidney (HEK) cells challenged with the same CB2-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors.

High throughput screening identifies ATP-competitive inhibitors of the NLRP1 inflammasome

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease.