Optimization Efforts for Identification of Novel Highly Potent Keap1-Nrf2 Protein-Protein Interaction Inhibitors.

In research focused on protein-protein interaction (PPI) inhibitors, the optimization process to achieve both high inhibitory activity and favorable physicochemical properties remains challenging. Our previous study reported the discovery of novel and bioavailable Keap1-Nrf2 PPI inhibitor 8 which exhibited moderate in vivo activity in rats. In this work, we present our subsequent efforts to optimize this compound. Two distinct approaches were employed, targeting high energy water molecules and Ser602 as "hot spots" from the anchor with good aqueous solubility, metabolic stability, and membrane permeability. Through ligand efficiency (LE)-guided exploration, we identified two novel inhibitors 22 and 33 with good pharmacokinetics (PK) profiles and more potent in vivo activities, which appear to be promising chemical probes among the existing inhibitors.

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.

Methyl and Fluorine Effects in Novel Orally Bioavailable Keap1-Nrf2 PPI Inhibitor.

Oxidative stress is one of the causes of progression of chronic kidney disease (CKD). Activation of the antioxidant protein regulator Nrf2 by inhibition of the Keap1-Nrf2 protein-protein interaction (PPI) is of interest as a potential treatment for CKD. We report the identification of the novel and weak PPI inhibitor 7 with good physical properties by a high throughput screening (HTS) campaign, followed by structural and computational analysis. The installation of only methyl and fluorine groups successfully provided the lead compound 25, which showed more than 400-fold stronger activity. Furthermore, these dramatic substituent effects can be explained by the analysis of using isothermal titration calorimetry (ITC). Thus, the resulting 25, which exhibited high oral absorption and durability, would be a CKD therapeutic agent because of the dose-dependent manner for up-regulation of the antioxidant protein heme oxigenase-1 (HO-1) in rat kidneys.

SAR study of small molecule inhibitors of the programmed cell death-1/programmed cell death-ligand 1 interaction.

The development of small molecule inhibitors of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) has drawn research interest for the treatment of cancer. Recently, we reported the discovery of a novel dimeric core small molecule PD-1/PD-L1 inhibitor. In an effort to discover more potent inhibitors, we further explored the dimeric core scaffold. Our investigations of the structure-activity-relationship revealed that introduction of lipophilic substituents onto one of the di-alkoxylated phenyl rings improved binding affinities to PD-L1, and inhibitory activities of PD-1/PD-L1 in cellular assays. Furthermore, conversion of the ether linker part to an olefin linker not only improved binding affinity but also led to slow dissociation binding kinetics. We also explored more potent, as well as downsized, scaffolds. Compounds bearing a linear chain in place of one of the di-alkoxylated phenyl rings exhibited good binding affinity with improved ligand efficiency (LE). Representative compounds demonstrated potent inhibitory activities of PD-1/PD-L1 in the submicromolar range in cellular assays as well as cellular function in the mixed lymphocyte reaction (MLR) assay with efficacy comparable to anti-PD-1 antibody. Our results provide applicable information for the design of more potent inhibitors targeting PD-1/PD-L1 pathway.

Symmetry-based ligand design and evaluation of small molecule inhibitors of programmed cell death-1/programmed death-ligand 1 interaction.

The development of small molecule inhibitors of PD-1/PD-L1 is eagerly anticipated for treatment of cancer. We focused on the symmetry of the ternary complex structure of reported small molecule ligands and hPD-L1 homodimers, and designed partially- or fully-symmetric compounds for more potent inhibitors. The design of the new compounds was guided by our hypothesis that the designed symmetric compound would induce a flip of sidechain of ATyr56 protein residue to form a new cavity. The designed compound 4 exhibited substantially increased binding affinity to hPD-L1, as well as PD-1/PD-L1 inhibitory activity in physiological conditions. Compound 4 also showed a dose-dependent increase in IFN-γ secretion levels in a mixed lymphocyte reaction assay. These results not only indicate the feasibility of targeting the PD-1/PD-L1 pathway with small molecules, but illustrate the applicability of the symmetry-based ligand design as an attractive methodology for targeting protein-protein interaction stabilizers.

Identification of a New Inhibitor That Stabilizes Interleukin-2-Inducible T-Cell Kinase in Its Inactive Conformation.

Interleukin-2-inducible T-cell kinase (ITK) plays an important role in T-cell signaling and is considered a promising drug target. As the ATP binding sites of protein kinases are highly conserved, the design of selective kinase inhibitors remains a challenge. Targeting inactive kinase conformations can address the issue of kinase inhibitor selectivity. It is important for selectivity considerations to identify compounds that stabilize inactive conformations from the primary screen hits. Here we screened a library of 390,000 compounds with an ADP-Glo assay using dephosphorylated ITK. After a surface plasmon resonance (SPR) assay was used to filter out promiscuous inhibitors, 105 hits were confirmed. Next, we used a fluorescent biosensor to enable the detection of conformational changes to identify inactive conformation inhibitors. A single-cysteine-substituted ITK mutant was labeled with acrylodan, and fluorescence emission was monitored. Using a fluorescent biosensor assay, we identified 34 inactive conformation inhibitors from SPR hits. Among them, one compound was bound to a site other than the ATP pocket and exhibited excellent selectivity against a kinase panel. Overall, (1) biochemical screening using dephosphorylated kinase, (2) hit confirmation by SPR assay, and (3) fluorescent biosensor assay that can distinguish inactive compounds provide a useful platform and offer opportunities to identify selective kinase inhibitors.

Identification of Novel Phospholipid Transfer Protein Inhibitors by High-Throughput Screening.

Atherogenesis has been recognized as a risk factor for lethal cardiovascular diseases. Plasma low-density lipoprotein levels are correlated to the occurrence of atherosclerosis, and their control is critical for both the prevention and treatment of these diseases. Phospholipid transfer protein (PLTP) is one of the key regulators of lipoprotein metabolism; PLTP-deficient mice exhibit decreased apolipoprotein B (apoB)-containing lipoprotein secretion and atherosclerosis, indicating the validity of PLTP as a promising therapeutic target. Here, we demonstrate a high-throughput screening (HTS) method to identify a novel chemotype of PLTP inhibitors. Instead of using recombinant proteins, we used human plasma as a source of enzymes in the first screening, so as to efficiently exclude promiscuous inhibitors. The selected compounds were further confirmed to target PLTP both biochemically and biophysically and were shown to inhibit apoB secretion from hepatic cells with no apparent toxicity. We believe that our approach is suitable for filtering out nonspecific inhibitors at an earlier stage of screening campaigns and that these compounds should have potential to be developed into drugs to treat dyslipidemia.

Identification of inactive conformation-selective interleukin-2-inducible T-cell kinase (ITK) inhibitors based on second-harmonic generation.

Many clinically approved protein kinase inhibitors stabilize an inactive conformation of their kinase target. Such inhibitors are generally highly selective compared to active conformation inhibitors, and consequently, general methods to identify inhibitors that stabilize an inactive conformation are much sought after. Here, we have applied a high-throughput, second-harmonic generation (SHG)-based conformational approach to identify small molecule stabilizers of the inactive conformation of interleukin-2-inducible T-cell kinase (ITK). A single-site cysteine mutant of the ITK kinase domain was created, labeled with an SHG-active dye, and tethered to a supported lipid bilayer membrane. Fourteen tool compounds, including stabilizers of the inactive and active conformations as well as nonbinders, were first examined for their effect on the conformation of the labeled ITK protein in the SHG assay. As a result, inactive conformation inhibitors were clearly distinguished from active conformation inhibitors by the intensity of SHG signal. Utilizing the SHG assay developed with the tool compounds described above, we identified the mechanism of action of 22 highly selective, inactive conformation inhibitors within a group of 105 small molecule inhibitors previously identified in a high-throughput biochemical screen. We describe here the first use of SHG for identifying and classifying inhibitors that stabilize an inactive vs. an active conformation of a protein kinase, without the need to determine costructures by X-ray crystallography. Our results suggest broad applicability to other proteins, particularly with single-site labels reporting on specific protein movements associated with selectivity.