Affinity map of bromodomain protein 4 (BRD4) interactions with the histone H4 tail and the small molecule inhibitor JQ1.

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histone tails via its tandem bromodomains BD1 and BD2 and forms a complex with the positive transcription elongation factor b, which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. We analyzed the binding of BRD4 BD1 and BD2 to different partners and showed that the strongest interactions took place with di- and tetra-acetylated peptides derived from the histone 4 N-terminal tail. We also found that several histone 4 residues neighboring the acetylated lysines significantly influenced binding. We generated 10 different BRD4 BD1 mutants and analyzed their affinities to acetylated histone tails and to the BET inhibitor JQ1 using several complementary biochemical and biophysical methods. The impact of these mutations was confirmed in a cellular environment. Altogether, the results show that Trp-81, Tyr-97, Asn-140, and Met-149 play similarly important roles in the recognition of acetylated histones and JQ1. Pro-82, Leu-94, Asp-145, and Ile-146 have a more differentiated role, suggesting that different kinds of interactions take place and that resistance mutations compatible with BRD4 function are possible. Our study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1 binding and may help in the design of new BET antagonists with improved pharmacological properties.

Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

BAY-8400: A Novel Potent and Selective DNA-PK Inhibitor which Shows Synergistic Efficacy in Combination with Targeted Alpha Therapies.

Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.

Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.

Structural Evidence for Isoform-Selective Allosteric Inhibition of Lactate Dehydrogenase A.

Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumors, thereby sustaining high glycolysis rates, tumor growth, and chemoresistance. High-throughput screening resulted in the identification of phthalimide and dibenzofuran derivatives as novel lactate dehydrogenase inhibitors, selectively inhibiting the activity of the LDHA isoenzyme. Cocrystallization experiments confirmed target engagement in addition to demonstrating binding to a novel allosteric binding site present in all four LDHA subunits of the LDH5 homotetramer.

The Novel ATR Inhibitor BAY 1895344 Is Efficacious as Monotherapy and Combined with DNA Damage-Inducing or Repair-Compromising Therapies in Preclinical Cancer Models.

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their efficacy.

Identification of Small Molecules that Modulate Mutant p53 Condensation.

Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.

Damage Incorporated: Discovery of the Potent, Highly Selective, Orally Available ATR Inhibitor BAY 1895344 with Favorable Pharmacokinetic Properties and Promising Efficacy in Monotherapy and in Combination Treatments in Preclinical Tumor Models.

The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).