Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk.

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.

Optimization of the efflux ratio and permeability of covalent irreversible BTK inhibitors.

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.

Discovery of potent, highly selective covalent irreversible BTK inhibitors from a fragment hit.

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.

Recent progress towards clinically relevant ATP-competitive Akt inhibitors.

The frequency of PI3K/Akt/mTOR (PAM) Pathway mutations in human cancers sparked interest to determine if the pathway is druggable. The modest clinical benefit observed with mTOR rapalogs (temsirolimus and everolimus) provided further motivation to identify additional nodes of pathway inhibition that lead to improved clinical benefit. Akt is a central signaling node of the PAM pathway and could be an ideal target for improved pathway inhibition. Furthermore, inhibitors of Akt may be especially beneficial in tumors with Akt1 mutations. Recently, multiple ATP-competitive Akt inhibitors have been identified and are currently in clinical development. This review details the medicinal chemistry efforts towards identification of these molecules, highlights relevant preclinical data supporting clinical evaluation, and summarizes current clinical development plans.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase.

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or "flip-flop" their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF(2)P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.