Hitoshi Iikura

Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.

Tumors with mutant RAS are often dependent on extracellular signal-regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Fluorine Scanning by Nonselective Fluorination: Enhancing Raf/MEK Inhibition while Keeping Physicochemical Properties

A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives, iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors.

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC50=8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED50=4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment.

Optimizing the Physicochemical Properties of Raf/MEK Inhibitors by Nitrogen Scanning

Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1 (CH5126766/RO5126766) was selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.

Abstract 3606: CH5126766 is a novel allosteric inhibitor of MEK that prevents its phosphorylation by RAF

Tumors with mutant BRAF or RAS are often dependent on the ERK signaling pathway. MEK and RAF inhibitors have significant antitumor activity in patients with BRAF mutant melanoma, but, thus far, MEK inhibitors have little activity in patients whose tumors contain mutant RAS. The effectiveness of the drugs seems to require near complete inhibition of ERK signaling. MEK inhibition has been shown to lead to relief of feedback inhibition of RAF and induction of MEK phosphorylation. It is possible that this induction attenuates inhibition of the pathway and reduces clinical activity. We have now isolated and characterized an allosteric inhibitor of MEK, CH5126766, with novel properties that prevent its induction of MEK phosphorylation. CH5126766 causes MEK to bind to RAF tightly and in a conformation in which it cannot be phosphorylated. MEK in this complex has low kinase activity and may act as a dominant negative inhibitor when bound to RAF. Compared to the MEK inhibitor PD0325901, CH5126766 inhibits ERK signaling output in cells more potently and has greater antitumor activity in the KRAS mutant colorectal cancer HCT116 grown as a murine xenograft. MEK inhibitors such as CH5126766 will inhibit feedback induction of MEK phosphorylation and this may allow more complete pathway inhibition and result in greater antitumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3606. doi:10.1158/1538-7445.AM2011-3606

Abstract 2789: Discovery of a novel specific MEK and Raf inhibitor, CH5126766 (RO5126766), hit to lead study of a unique scaffold for kinase inhibitor to a clinical compound

Background: Among tumor signaling pathways, the most frequently dysregulated in human cancer is Ras-Raf-MEK axis, and therefore allosteric MEK inhibitors and ATP competitive Raf inhibitors are being tested in clinical trials. However, these inhibitors result in marked activation of upstream signal of MEK in tumor cells, especially with mutant K-ras. This feedback activation or activation of Raf would attenuate the anticancer effects of these inhibitors. One of the strategies to prevent this attenuation will be blocking upstream signal concurrent with MEK inhibition. To validate the hypothesis, we executed a hit to lead study of our HTS hit showing MEK/Raf dual inhibition. Methods and Results: To increase direct MEK inhibitory activity (by binding to MEK) of the HTS hit coumarin compound and indirect Raf inhibitory activity (by enhancement of the binding between B-Raf /C-Raf and MEK1), introduction of a sulfamide group afforded the best result, compared with similar functional groups such as urea, amide and sulfonamide. Fluorine-scan (C-H/C-F substitutions) and nitrogen-scan (CH/N substitutions) worked efficient to increase both MEK and Raf inhibitory activities and optimize physicochemical properties (water solubility, hERG inhibition, CYP inhibitions etc). These three key transformations afforded CH5126766, which has MEK1 (IC50 = 160 nM) and B-Raf (IC50 = 19 nM) inhibitions and excellent PK profiles (mouse BA 93%, CL 1.1 mL/min/kg, rat BA 66%, CL 0.7 mL/min/kg, monkey BA 82%, CL 0.1 mL/min/kg). The compound showed potent antitumor activity in human cancer xenograft model (HCT116) by once daily oral administration (128% of tumor growth inhibition at a 1 mg/kg dose). Conclusion: SAR study of our novel type molecule (MEK and Raf dual inhibitor) affords identification of CH5126766. It showed strong antitumor efficacy in vivo, good PK in three animal species and good safety profiles. Thus, CH5126766 is now under clinical investigation with solid tumor patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2789. doi:10.1158/1538-7445.AM2011-2789