Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors.

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

Optimization of tetrahydronaphthalene inhibitors of Raf with selectivity over hERG.

Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.

A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts.

Objective. Use of tyramide signal amplification (TSA) to detect autophagy biomarkers in formalin fixed and paraffin embedded (FFPE) xenograft tissue. Materials and Methods. Autophagy marker regulation was studied in xenograft tissues using Amp HQ IHC and standard IHC methods. Results. The data demonstrate the feasibility of using high sensitivity TSA IHC assays to measure low abundant autophagy markers in FFPE xenograft tissue.

Antitumor activity of the selective pan-RAF inhibitor TAK-632 in BRAF inhibitor-resistant melanoma.

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors.

Design and optimization of potent and orally bioavailable tetrahydronaphthalene Raf inhibitors.

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.