Potent BRAFV600E Inhibitors Research Articles

Abstract 598: HSK42360: A potent, brain permeable, BRAF paradox breaker for the treatment of BRAF-driven cancers

Abstract Mutations in the BRAF gene are among the most common identified in human cancers approximately 7% of all cancers evaluated, including 70% of melanomas, 50% of thyroid-like cancers, 10% to 20% of colorectal cancers, 13% of serous ovarian cancers, 3-7% of non-small-cell lung cancers, and most recently, hairy cell leukemia. Approved BRAF inhibitors (or +MEKi) against BRAFV600E/K improved initial clinical efficacy. However intrinsic and acquired resistance often arise through BRAF paradoxical activation, limiting durable responses to current BRAF inhibitors. In addition, Approved BRAFi responds poorly to patients with BRAF mutant gliomas or brain metastases, mainly due to limited brain permeability. More effective therapeutic options are therefore needed. To address this clinical challenge, we developed a potent, brain-penetrating paradox breaker HSK42360, as a promising therapy for patients with BRAFV600E mutation, progression or brain metastases following treatment with approved BRAFi or BRAFi/MEKi therapies. HSK42360 is a potent BRAFV600E inhibitor with IC50 value of 5 nM at enzymatic level. Further characterization showed that HSK42360 did not induce homodimer or heterodimer of CRAF with CRAF or BRAF and thereby blocked reactivation of the MAPK pathway in HCT116 cells, whereas Dabrafenib significantly induced RAF dimerization and MAPK reactivation. HSK42360 exhibited significant anti-proliferation activity against multiple tumor cell lines with BRAFV600E, including A375 (melanoma), DU4475 (breast cancer), and COLO205 (colon cancer), but was highly selective for BRAF wild-type cells. HSK42360 was administered orally once daily and showed broad-spectrum antitumor activity in the COLO205 and A375 CDX models. Immunohistochemistry showed a close correlation between the inhibition of tumor growth and ERK1/2 phosphorylation in A375 CDX tumor tissues. To explored the impact of HSK42360 in a brain micro-metastasis model aimed at mimicking early CNS metastatic spread, tumor progression was monitored through BLI (full name) imaging, which revealed that HSK42360 was highly brain penetrant with Kp,uu greater than 1 and triggered potent antitumor activity and prolonged survival in brain metastatic models of melanoma. We next tested the hypothesis that HSK42360 could retain activity in models presenting RAF dimer-driven resistance. Surprisingly, HSK42360 demonstrated promising antitumor effects in the first gen-resistant NRASmA375(NRASQ61K, BRAFV600E) CDX model, supporting its use as a follow-on therapy for current BRAFi resistance. Collectively, these preclinical studies validated that HSK42360 is a novel brain-permeable BRAF paradox breaker that exhibits outstanding antitumor activity in brain metastasis and RAF dimer-driven resistance. Citation Format: Shidong Gao, Yangguang Li, Ju Wang, Pingming Tang, Zongjun Shi, Yao Li, Pangke Yan. HSK42360: A potent, brain permeable, BRAF paradox breaker for the treatment of BRAF-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 598.

Rafoxanide, an Organohalogen Drug, Triggers Apoptosis and Cell Cycle Arrest in Multiple Myeloma By Enhancing DNA Damage Responses and Suppressing the p38 MAPK Pathway

Rafoxanide is used in veterinary medicine for the treatment of fascioliasis. We previously repositioned the drug as the inhibitor of B-Raf V600E, but its anti-tumor effect in human cancer has never been reported. In this study, we investigated the effects of rafoxanide in multiple myeloma (MM) in vitro and in vivo. We found that rafoxanide inhibited cell proliferation and overcame the protective effect of the bone marrow (BM) microenvironment on MM cells. Rafoxanide induced cell apoptosis by reducing mitochondrial membrane potential (MMP) and regulating the caspase pathway, while having no apparent toxic effect on normal cells. Rafoxanide also inhibited DNA synthesis and caused cell cycle arrest by regulating the cdc25A-degradation pathway. In addition, rafoxanide enhanced the DNA damage response by up-regulating the expression of γ-H2AX. Importantly, as a potent B-Raf V600E inhibitor, rafoxanide could suppress activation of the p38 MAPK pathway. Rafoxanide treatment inhibited tumor growth, with no significant side effects, in an MM mouse xenograft model. Finally, combination of rafoxanide with bortezomib or lenalidomide significantly induced synergistic cytotoxicity in MM cells. Collectively, our results may provide a rationale for use of this drug in MM treatment. This study was supported by grants from the National Natural Science Foundation of China (Nos. 81570190; 81670194 and 81529001). DisclosuresNo relevant conflicts of interest to declare.

Docking-based structural splicing and reassembly strategy to develop novel deazapurine derivatives as potent B-RafV600E inhibitors.

The mutation of B-RafV600E is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched as drugs for treating unresectable melanoma, demonstrating that B-RafV600E is an ideal drug target. This study focused on developing novel B-RafV600E inhibitors as drug leads against various cancers with B-RafV600E mutation. Using molecular modeling approaches, 200 blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular structures of potential inhibitors of B-RafV600E were then obtained by fragment reassembly followed by docking to predict the bioactivity of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify potent B-RafV600E inhibitors. A highly potent fragment binding to the hinge area of B-RafV600E was identified via a docking-based structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were predicted to be as strong as marketed B-RafV600E inhibitors. Biological evaluation revealed that compound 1m is a potent B-RafV600E inhibitor with an IC50 value of 0.05 μmol/L, which was lower than that of vemurafenib (0.13 μmol/L). Moreover, the selectivity of 1m against B-RafWT was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic stability in in vitro assays. Thus, a highly potent and selective B-RafV600E inhibitor was designed via a docking-based structural splicing and reassembly strategy and was validated by medicinal synthesis and biological evaluation.

Computational investigation on inhibition mechanism of BRAFV600E by Vemurafenib (PLX4032) and its analogue PLX4720

BRAFV600E small-molecule inhibitors have recently received increasing attention as very effective agents for the therapy of cancer, especially melanoma. In this current work, a computational investigation was performed to investigate the interaction details of BRAFV600E kinase with inhibitors Vemurafenib (PLX4032) and its analog PLX4720. Binding free energy calculations based on molecular dynamics simulations reveal that ligands PLX4032 and PLX4720 bind to and stabilize the DFG-in conformation of BRAFV600E. Component analysis of binding free energy revealed that vdWaals interactions play a dominating effect on BRAFV600E inhibition. Furthermore, the per-residue binding free energy decomposition revealed that the most favorable contribution came from Ile463, Val471, Ala481, Lys483, Leu514, Ile527, Thr529, Gln530, Trp531, Cys532, Phe583, Gly593, and Asp594. These results agree well with experimental data, which provide valuable resources for understanding the inhibition mechanism of BRAFV600E by PLX4032 and PLX4720 and clues for the design of novel potent BRAFV600E inhibitors.

Determination of A Novel Selective B-RafV600E Inhibitor (LXK4) in Dog Plasma by HPLC–MS/MS and its Application in a Pharmacokinetic Study

A rapid and sensitive HPLC–MS/MS assay was established and validated to quantify LXK4, a novel potent B-RafV600E inhibitor in dog plasma. The samples prepared by liquid–liquid extraction using ethyl acetate were separated on a Phenomenex C8 column with mobile phase of water–methanol. LXK4 and IS were all detected by monitoring the precursor → product ion transition of m/z 413.4 → 345.4 and 401.3 → 333.3, respectively, using multiple reaction monitoring (MRM) scan mode with positive ionization mode. The liner calibration curves for LXK4 in dog plasma ranged from 0.5 to 1000 ng mL−1 with the lower limit of quantification of 0.5 ng mL−1. The intra- and inter-day precisions were lower than 12.0%, and the accuracy ranged from −4.6 to 9.5%. The validated assay was successfully used as a new approach to study the pharmacokinetics of LXK4 in beagle dogs and the oral bioavailability of LXK4 was determined to be 30.5%.

Photoinduced Conversion of Antimelanoma Agent Dabrafenib to a Novel Fluorescent BRAFV600E Inhibitor.

Dabrafenib (Tafinlar) was approved in 2013 by the FDA as a selective single agent treatment for patients with BRAFV600E mutation-positive advanced melanoma. One year later, a combination of dabrafenib and trametinib was used for treatment of BRAFV600E/K mutant metastatic melanoma. In the present study, we report on hitherto not described photosensitivity of dabrafenib both in organic and aqueous media. The half-lives for dabrafenib degradation were determined. Moreover, we revealed photoinduced chemical conversion of dabrafenib to its planar fluorescent derivative dabrafenib_photo 2. This novel compound could be isolated and biologically characterized in vitro. Both enzymatic and cellular assays proved that 2 is still a potent BRAFV600E inhibitor. The intracellular formation of 2 from dabrafenib upon ultraviolet irradiation is shown. The herein presented findings should be taken in account when handling dabrafenib both in preclinical research and in clinical applications.

Design, synthesis and biological evaluation of novel benzo-α-pyrone containing piperazine derivatives as potential BRAFV600E inhibitors

The increasingly acquired resistance to vemurafenib and side effects of known inhibitors motivate the search for new and more effective anti-melanoma drugs. In this Letter, virtual screening and scaffold growth were combined together to achieve new molecules as BRAFV600E inhibitors. Along with docking simulation, a primary screen in vitro was performed to filter the modifications for the lead compound, which was then substituted, synthesized and evaluated for their inhibitory activity against BRAFV600E and several melanoma cell lines. Out of the obtained compounds, derivative 3l was identified as a potent BRAFV600E inhibitor and exerted an anticancer effect through BRAFV600E inhibition. The following biological evaluation assays confirmed that 3l could induce cell apoptosis and marked DNA fragmentation. Furthermore, 3l could arrest the cell cycle at the G0/G1 phase in melanoma cells. The docking simulation displayed that 3l could tightly bind with the crystal structure of BRAFV600E at the active site. Overall, the biological profile of 3l suggests that this compound may be developed as a potential anticancer agent.

Repositioning organohalogen drugs: a case study for identification of potent B-Raf V600E inhibitors via docking and bioassay.

Drug repositioning has been attracting increasingly attention for its advantages of reducing costs and risks. Statistics showed that around one quarter of the marketed drugs are organohalogens. However, no study has been reported, to the best of our knowledge, to aim at efficiently repositioning organohalogen drugs, which may be attributed to the lack of accurate halogen bonding scoring function. Here, we present a study to show that two organohalogen drugs were successfully repositioned as potent B-Raf V600E inhibitors via molecular docking with halogen bonding scoring function, namely D3DOCKxb developed in our lab, and bioassay. After virtual screening by D3DOCKxb against the database CMC (Comprehensive Medicinal Chemistry), 3 organohalogen drugs that were predicted to form strong halogen bonding with B-Raf V600E were purchased and tested with ELISA-based assay. In the end, 2 of them, rafoxanide and closantel, were identified as potent inhibitors with IC50 values of 0.07 μM and 1.90 μM, respectively, which are comparable to that of vemurafenib (IC50: 0.17 μM), a marketed drug targeting B-Raf V600E. Single point mutagenesis experiments confirmed the conformations predicted by D3DOCKxb. And comparison experiment revealed that halogen bonding scoring function is essential for repositioning those drugs with heavy halogen atoms in their molecular structures.

Identification and synthesis of N-(thiophen-2-yl) benzamide derivatives as BRAFV600E inhibitors

The V600E BRAF kinase mutation, which activates the downstream MAPK signaling pathway, commonly occurs in about 8% of all human malignancies and about 50% of all melanomas. In this study, we employed virtual screening and chemical synthesis to identify a series of N-(thiophen-2-yl) benzamide derivatives as potent BRAFV600E inhibitors. Structure–activity relationship studies of these derivatives revealed that compounds b40 and b47 are the two most potent BRAFV600E inhibitors in this series.