Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues.

Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.

Fragment-Based Discovery of Novel VE-PTP Inhibitors Using Orthogonal Biophysical Techniques.

Tyrosine phosphorylation is an essential post-translational modification that regulates various biological events and is implicated in many diseases including cancer and atherosclerosis. Vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays an important role in vascular homeostasis and angiogenesis, is therefore an attractive drug target for these diseases. However, there are still no drugs targeting PTP including VE-PTP. In this paper, we report the discovery of a novel VE-PTP inhibitor, Cpd-2, by fragment-based screening combining various biophysical techniques. Cpd-2 is the first VE-PTP inhibitor with a weakly acidic structure and high selectivity, unlike known strongly acidic inhibitors. We believe that this compound represents a new possibility for the development of bioavailable VE-PTP inhibitors.

The discovery of 3,3-dimethyl-1,2,3,4-tetrahydroquinoxaline-1-carboxamides as AMPD2 inhibitors with a novel mechanism of action.

AMP deaminase 2 (AMPD2) has been thought to play an important role in energy homeostasis and immuno-oncology, while selective AMPD2 inhibitors are highly demanded to clarify the physiological function of AMPD2. In this report, we describe selective AMPD2 inhibitors inducing allosteric modulation. Based on hypothesis that compounds that exhibit increased inhibition by preincubation would cause conformational change of the enzyme, starting from HTS hit compound 4, we discovered compound 8 through the SAR study. From X-ray structural information of 8, this chemical series has a novel mechanism of action that changes the substrate pocket to prevent AMP from binding. Further elaboration of compound 8 led to the tool compound 21 which exhibited potent inhibitory activity of AMPD2 in ex vivo evaluation of mouse liver.

Structure-based drug design of novel and highly potent pyruvate dehydrogenase kinase inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) are fascinating drug targets for numerous diseases, including diabetes and cancers. In this report, we describe the result of our structure-based drug design from tricyclic lead compounds that led to the discovery of highly potent PDHK2 and PDHK4 dual inhibitors in enzymatic assay. The C3-position of the tricyclic core was explored, and the PDHK2 X-ray structure with a representative compound revealed a novel ATP lid conformation in which the phenyl ring of Phe326 mediated the interaction of the Arg258 sidechain and the compound. Compounds with amide linkers were designed to release the ATP lid by forming an intramolecular pi-pi interaction, and these compounds showed single-digit nM IC50 values in an enzymatic assay. We also explored the C4-position of the tricyclic core to reproduce the interaction observed with the C3-position substitution, and the pyrrolidine compound showed the same level of IC50 values. By optimizing an interaction with the Asn255 sidechain through a docking simulation, compounds with 2-carboxy pyrrole moiety also showed single-digit nM IC50 values without having a cation-pi interaction with the Arg258 sidechain.

Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

Ternary crystal structure of human RORγ ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction.

Retinoic acid-related orphan receptor gamma (RORγ) plays pivotal roles in autoimmune diseases by controlling the lineage of interleukin 17 (IL-17)-producing CD4+ T cells (Th17 cells). Structure-based drug design has proven fruitful in the development of inhibitors targeting the ligand binding domain (LBD) of RORγ. Here, we present the crystal structure of a novel RORγ inhibitor co-complex, in the presence of a corepressor (CoR) peptide. This ternary complex with compound T reveals the structural basis for an inhibitory mechanism different from the previously reported inverse agonist. Compared to the inverse agonist, compound T induces about 2 Šshift of helix 5 (H5) backbone and side-chain conformational changes of Met365 on H5. These conformational changes correlate to reduced CoR peptide binding to RORγ-LBD in the presence of compound T, which suggests that the shift of H5 is responsible. This crystal structure analysis will provide useful information for the development of novel and efficacious drugs for autoimmune disorders.