The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Discovery of KIN-3248, An Irreversible, Next Generation FGFR Inhibitor for the Treatment of Advanced Tumors Harboring FGFR2 and/or FGFR3 Gene Alterations.

Fibroblast growth factor receptor (FGFR) alterations are present as oncogenic drivers and bypass mechanisms in many forms of cancer. These alterations can include fusions, amplifications, rearrangements, and mutations. Acquired drug resistance to current FGFR inhibitors often results in disease progression and unfavorable outcomes for patients. Genomic profiling of tumors refractory to current FGFR inhibitors in the clinic has revealed several acquired driver alterations that could be the target of next generation therapeutics. Herein, we describe how structure-based drug design (SBDD) was used to enable the discovery of the potent and kinome selective pan-FGFR inhibitor KIN-3248, which is active against many acquired resistance mutations. KIN-3248 is currently in phase I clinical development for the treatment of advanced tumors harboring FGFR2 and/or FGFR3 gene alterations.

Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1).

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

Structure-based design and discovery of potent and selective lysine-specific demethylase 1 (LSD1) inhibitors.

The histone demethylase LSD1 is a key enzyme in the epigenetic regulation of gene transcription. Here we present our efforts to discover small molecule reversible inhibitors of LSD1 as an attractive approach to treat hematologic malignancies and certain solid tumors. Using structure-based drug design, we designed and synthesized a novel series of heteroaromatic imidazole inhibitors that demonstrate potent inhibition of the demethylase activity and low nanomolar cell-based activity. This novel LSD1 inhibitor series was further optimized by attenuating the hERG inhibition and improving oral bioavailability.

KDM4 Inhibition Targets Breast Cancer Stem-like Cells.

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Cancer Res; 77(21); 5900-12. ©2017 AACR.

Design of KDM4 Inhibitors with Antiproliferative Effects in Cancer Models.

Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound 6 (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.

Lsd1 prevents age-programed loss of beige adipocytes.

Aging is accompanied by major changes in adipose tissue distribution and function. In particular, with time, thermogenic-competent beige adipocytes progressively gain a white adipocyte morphology. However, the mechanisms controlling the age-related transition of beige adipocytes to white adipocytes remain unclear. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser enzyme positively regulating differentiation and function of adipocytes. Here we show that Lsd1 levels decrease in aging inguinal white adipose tissue concomitantly with beige fat cell decline. Accordingly, adipocyte-specific increase of Lsd1 expression is sufficient to rescue the age-related transition of beige adipocytes to white adipocytes in vivo, whereas loss of Lsd1 precipitates it. Lsd1 maintains beige adipocytes by controlling the expression of peroxisome proliferator-activated receptor α (Ppara), and treatment with a Ppara agonist is sufficient to rescue the loss of beige adipocytes caused by Lsd1 ablation. In summary, our data provide insights into the mechanism controlling the age-related beige-to-white adipocyte transition and identify Lsd1 as a regulator of beige fat cell maintenance.

Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4.

Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.

Lsd1 Ablation Triggers Metabolic Reprogramming of Brown Adipose Tissue.

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT-selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1-deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT.

Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton's Tyrosine Kinase.

The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton's tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

Design and synthesis of orally available MEK inhibitors with potent in vivo antitumor efficacy.

The structure-based design, synthesis, and biological evaluation of two novel series of potent and selective MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic dihydroindolone core provided compounds with high potency and good drug-like pharmaceutical properties. Further scaffold modification afforded a series of dihydroindolizinone inhibitors, including an orally available advanced preclinical MEK inhibitor with potent in vivo antitumor efficacy.

Discovery of TAK-733, a potent and selective MEK allosteric site inhibitor for the treatment of cancer.

A novel 5-phenylamino-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione series of MEK inhibitors has been developed using structure-based drug design. Lead optimization of this series led to the discovery of TAK-733. This was advanced to Phase I clinical studies for cancer treatment.

Structure-based design and synthesis of pyrrole derivatives as MEK inhibitors.

A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design. Optimization of the series led to the identification of potent inhibitors with good pharmaceutical properties.