Identification and Characterization of Potential BRDT(1) Inhibitors by Fragment-Based Screening Using Differential Screening Fluorimetry and Orthogonal Techniques

Abstract

Almost 60 years after the advent of the female oral contraceptive pill, there are now a plethora of hormonal contraceptive options for women. Despite all of these available methods, 43% of pregnancies in the United states from 2011 to 2015 were unintended. Contraceptive methods for men are limited to condoms and vasectomies, neither of which offer complete protection from unintended pregnancies. Development of effective, reversible, nonhormonal male contraceptives would be a revolution in the area of contraception, complementing currently available methods and offering a new alternative to men. Epigenetics is a rising area of interest, concerned with the differential expression of genes caused by post-translational modification of proteins. Acetylation of lysine residues is regulated by several groups of proteins, including the bromodomains, responsible for recognizing the acetyl modifications and recruiting other effector molecules to impact chromatin remodeling, transcriptional control, and DNA repair. The bromodomain and extra terminal (BET) family of bromodomains has been implicated in many critical processes (such as cell cycle progression, transcriptional elongation) and disease states, including aggressive leukemias, obesity, acute inflammation, and NUT midline carcinoma. One BET protein, BRDT, is expressed exclusively in the testes and is responsible for regulating spermatogenesis. Knockout models of BRDT yield healthy, normal male offspring that are completely infertile. Studies using small molecule inhibitors of BET proteins have shown that BRDT inhibition causes a reversible infertility, making BRDT an attractive target for a non-hormonal male contraceptive. A fragment-based drug design strategy was employed to identify potential BRDT inhibitor leads. Differential scanning fluorimetry (DSF) was optimized for BRDT and used to screen a fragment library. An initial collection of 34 positive- and negative-shifting fragment hits were identified and confirmed from fresh powder by the DSF assay. Protein-observed 19F NMR (PrOF-NMR), fluorescence polarization (FP), amplified luminescent proximity homogenous assay screen (AlphaScreen), and x-ray crystallography were all part of the orthogonal screening cascade used to confirm BRDT binding. In total, 16 fragments were confirmed as ligands of BRDT, including a novel 1,5-benzodiazepinone scaffold. A number of these hits were pursued by our lab as lead molecules. In particular, and indanone scaffold was selected for further investigation. Analogs of the hit fragment were designed to develop structure-activity relationships of the chemotype; the indanone headgroup, linker composition, and substitution of a terminal phenyl group were studied. The analogs were tested by DSF, PrOF-NMR, FP, and AlphaScreen, with the most potent compounds submitted for crystallography experiments. The DSF assay developed for fragment screening was also used to test a number of other compounds for BRDT binding activity, including a pyridone scaffold identified by the DSF screen, several series based on kinase inhibitors shown to bind BET proteins, and a series based on a virtual high throughput screening hit.