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The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are responsive to RAF inhibitors such as dabrafenib and vemurafenib, these drugs are ineffective in RAS mutant cancers and tumors expressing other RAF mutations. CRAF kinase functions as a critical effector in mutant RAS and Class II/III BRAF mutant tumors and plays a role in feedback-mediated pathway reactivation following MEK inhibition. Thus, selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS and BRAF signaling and in inhibiting feedback-mediated activation in combination with a MEK inhibitor. LXH254 is a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar concentrations with a high degree of selectivity against a panel of 456 human kinases and in cell-based assays. LXH254 not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling due to its ability to inhibit both RAF monomers and dimers with similar potencies. LXH254 is orally bioavailable, demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. LXH254 represents a next generation RAF inhibitor that is differentiated from other RAF inhibitors in this class due to the high degree of selectivity. In preclinical efficacy and toxicology studies, LXH254 demonstrated a relatively wide therapeutic index which should enable effective interrogation of RAF inhibition in patients with decreased risk for off-target toxicity. LXH254 is currently in a Phase I trial in patients with solid tumors expressing MAPK pathway mutations. Citation Format: Darrin D. Stuart, Wenlin Shao, Yuji Mishina, Yun Feng, Giordano Caponigro, Vesselina G. Cooke, Stacey Rivera, Fang Shen, Joshua Korn, Lesley A. Mathews Griner, Giselle Nishiguchi, Benjamin Taft, Lifeng Wan, Sharadha Subramanian, Yan Lou, Lina Setti, Matthew Burger, Victor Tamez, Alice Rico, Robert Aversa, John Tellew, Jacob R. Haling, Valery Polyakov, Amy Lambert, Richard Zang, Ann Van Abbema, Mohamad Hekmat-Nejad, Payman Amiri, Mallika Singh, Nicholas Keen, Michael P. Dillon, Emma Lees, William R. Sellers, Savithri Ramurthy. Pharmacological profile and anti-tumor properties of LXH254, a highly selective RAF kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT01-04.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor–derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537–48. ©2018 AACR.

RAS oncogenes have been implicated in30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.

The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes in the pathway, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are effectively treated with existing RAF inhibitors, RAS mutant cancers and tumors expressing atypical BRAF mutants remain an unmet medical need. Emerging biology has demonstrated that the CRAF kinase functions as a critical mediator of mutant KRAS-driven cell proliferation and tumor development. CRAF was also shown to be the mediator of feedback-mediated pathway reactivation following MEK inhibitor treatment in KRAS mutant cancers. Hence selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS-driven tumorigenesis and in alleviating feedback activation. We have developed a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar IC50 values in biochemical assays with high selectivity profile against a panel of 456 human kinases. The inhibitor not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling with minimum paradoxical activation, likely due to its activity in inhibiting both RAF monomers and dimers with similar potencies. Correspondingly, profiling data of the inhibitor in a panel of 480 human cancer cell lines shows that it has higher antitumor activities in cell lines harboring BRAF or RAS mutations as compared to those that are wild-type. The inhibitor is orally bioavailable, it demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models that have BRAF, NRAS or KRAS mutations with good tolerability. Thus, we have developed a next generation RAF inhibitor with unique biochemical and cellular properties that enables its antitumor activities in RAS mutant tumors. Citation Format: Wenlin Shao, Yuji Mishina, Yun Feng, Giordano Caponigro, Savithri Ramurthy, Vesselina Cooke, Lesley Griner, Gisele Nishiguchi, Alice Rico, Ben Taft, Matthew Burger, Huw Tanner, Valery Polyakov, Brent Appleton, John Tellew, Richard Zang, Mohammad Hekmat-Nejad, Payman Amiri, Mallika Singh, Darrin Stuart. Development of a highly selective B/CRAF kinase inhibitor that exhibits antitumor activities in RAS and BRAF mutant tumors with minimal paradoxical activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 330.

The immunoglobulin E (IgE) response is generally considered an essential component of the host defense against parasitic helminths such as Schistosoma mansoni. In contrast, work on antischistosome vaccines suggests that interferon gamma (IFN-gamma) is the critical immune mediator for vaccine-induced immunity to the parasite. In this study, the total IgE response to a primary S. mansoni infection was suppressed by anti-IgE treatment in both normal mice and in mice with defective IFN genes (gene knockout [GKO]). Reduction of the IgE response resulted in decreased worm burden and a decrease in the number of eggs produced per worm in both normal and GKO mice. Whereas anti-IgE treatment also resulted in reduced hepatosplenomegaly, granulomas around existing schistosome eggs showed normal cellularity. Serum interleukin 4 levels fell in response to the reduction in serum IgE as well. The data suggest that IgE plays a detrimental, rather than beneficial, role for the host in schistosomiasis. Furthermore, the absence of IFN-gamma was found to be of little consequence to the host-response to adults or eggs in a primary schistosome infection.

Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1β converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1.

The error is on page 990 in Table 1. Compound 11 should be 3-OCF3, and compound 16 should be 4-OCF3.

Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed "ducts" which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite.

The cercarial acetabular gland proteinase of Schistosomatium douthitti, an agent of ‘swimmer's itch’, has been identified and characterized. Like the corresponding proteinase of Schistosoma mansoni, it has significant elastase activity and can degrade a model of dermal extracellular matrix. However, unlike the S. mansoni enzyme, it has a higher molecular weight (50 000 versus 30 000), is of a different proteinase class (metallo versus serine), and has no significant primary structure homology to the S. mansoni proteinase. While these findings indicate that the failure of S. douthitti to produce chronic schistosomiasis in humans is not due to its lacking, or having a less potent ‘penetration proteinase’ than S. mansoni, the proteolytic enzymes are sufficiently different to support the hypothesis that the Schistosomatium line diverged quite early from the main branch of Schistosoma evolution.