Design, synthesis, and biological evaluation of selective covalent inhibitors of FGFR4.

Aberrant signaling via fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) has been identified as a driver of tumorigenesis and the development of many solid tumors, making FGFR4 is a promising target for anticancer therapy. Herein, we designed and synthesized a series of bis-acrylamide covalent FGFR4 inhibitors and evaluated their inhibitory activity against FGFRs, FGFR4 mutants, and their antitumor activity. CXF-007, verified by mass spectrometry and crystal structures to form covalent bonds with Cys552 of FGFR4 and Cys488 of FGFR1, exhibited stronger selectivity and potent inhibitory activity for FGFR4 and FGFR4 cysteine mutants. Moreover, CXF-007 exhibited significant antitumor activity in hepatocellular carcinoma cell lines and breast cancer cell lines through sustained inhibition of the FGFR4 signaling pathway. In summary, our study highlights a novel covalent FGFR4 inhibitor, CXF-007, which has the potential to overcome drug-induced FGFR4 mutations and might provide a new strategy for future anticancer drug discovery.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

Structural basis and selectivity of sulfatinib binding to FGFR and CSF-1R.

Acquired drug resistance poses a challenge for single-target FGFR inhibitors, leading to the development of dual- or multi-target FGFR inhibitors. Sulfatinib is a multi-target kinase inhibitor for treating neuroendocrine tumors, selectively targeting FGFR1/CSF-1R. To elucidate the molecular mechanisms behind its binding and kinase selectivity, we determined the crystal structures of sulfatinib with FGFR1/CSF-1R. The results reveal common structural features and distinct conformational adaptability of sulfatinib in response to FGFR1/CSF-1R binding. Further biochemical and structural analyses disclose sensitivity of sulfatinib to FGFR/CSF-1R gatekeeper mutations. The insensitivity of sulfatinib to FGFR gatekeeper mutations highlights the indispensable interactions with the hydrophobic pocket for FGFR selectivity, whereas the rotatory flexibility may enable sulfatinib to overcome CSF-1RT663I. This study not only sheds light on the structural basis governing sulfatinib's FGFR/CSF-1R inhibition, but also provides valuable insights into the rational design of dual- or multi-target FGFR inhibitors with selectivity for CSF-1R and sensitivity to gatekeeper mutations.

Discovery of D6808, a Highly Selective and Potent Macrocyclic c-Met Inhibitor for Gastric Cancer Harboring MET Gene Alteration Treatment.

MET alterations have been validated as a driven factor in NSCLC and gastric cancers. The c-Met inhibitors, capmatinib, tepotinib, and savolitinib, are only approved for the treatment of NSCLC harboring exon 14 skipping mutant MET. We used a molecular hybridization in conjunction with macrocyclization strategy for structural optimization to obtain a series of 2-(2-(quinolin-6-yl)ethyl)pyridazin-3(2H)-one derivatives as new c-Met inhibitors. One of the macrocyclic compounds, D6808, potently inhibited c-Met kinase and MET-amplified Hs746T gastric cancer cells with IC50 values of 2.9 and 0.7 nM, respectively. It also strongly suppressed Ba/F3-Tpr-Met cells harboring resistance-relevant mutations (F1200L/M1250T/H1094Y/F1200I/L1195V) with IC50 values of 4.2, 3.2, 1.0, 39.0, and 33.4 nM, respectively. Furthermore, D6808 exhibited extraordinary target specificity in a Kinome profiling against 373 wild-type kinases and served as a promising macrocycle-based compound for further anticancer drug development.

Site-specific N-terminal PEGylation-based controlled release of biotherapeutics: An application for GLP-1 delivery to improve pharmacokinetics and prolong hypoglycemic effects.

PEGylation is a well-established technology for half-life extension in drug delivery. In this study, we aimed to develop a site-specific N-terminal PEGylation for biotherapeutics to achieve controlled release, using GLP-1 as a model. An additional threonine was introduced at N-terminal GLP-1. Followed by periodate oxidation, hydrazide-based PEGylation was achieved in a site-selective manner under reductive condition. Two homogenous monovalent mPEG5k-GLP-1 (peptide 4) and mPEG20k-GLP-1 (peptide 5) were successfully constructed. After PEGylation, the degradation by DPP-IV and rat plasma was obviously reduced. Their pharmacokinetic performances were enhanced at the expense of impaired GLP-1R stimulating potency, and their hypoglycemic effects were improved in different degrees. Compared with conventional strategies, this approach is devoid of the restriction and alteration of native peptide sequences, and can produce utterly homogenous conjugates with excellent selectivity and efficiency. It provides a practical controlled release approach for peptides by site-specific modification to achieve better pharmacological and therapeutic properties.

Design, Synthesis, and Biological Evaluation of Aminoindazole Derivatives as Highly Selective Covalent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR4.

Aberrant FGF19/FGFR4 signaling has been shown to be an oncogenic driver of growth and survival in human hepatocellular carcinoma (HCC) with several pan-FGFR inhibitors and FGFR4-selective inhibitors currently being evaluated in the clinic. However, FGFR4 gatekeeper mutation induced acquired resistance remains an unmet clinical challenge for HCC treatment. Thus, a series of aminoindazole derivatives were designed and synthesized as new irreversible inhibitors of wild-type and gatekeeper mutant FGFR4. One representative compound (7v) exhibited excellent potency against FGFR4, FGFR4V550L, and FGFR4V550M with nanomolar activity in both the biochemical and cellular assays while sparing FGFR1/2/3. While compound 7v demonstrated modest in vivo antitumor efficacy in nude mice bearing the Huh-7 xenograft model consistent with its unfavorable pharmacokinetic properties, it provides a promising new starting point for future drug discovery combating FGFR4 gatekeeper mediated resistance in HCC patients.

Structure-based design of a dual-warhead covalent inhibitor of FGFR4.

The fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) signaling pathways play critical roles in a variety of cancers, such as hepatocellular carcinoma (HCC). FGFR4 is recognized as a promising target to treat HCC. Currently, all FGFR covalent inhibitors target one of the two cysteines (Cys477 and Cys552). Here, we designed and synthesized a dual-warhead covalent FGFR4 inhibitor, CXF-009, targeting Cys477 and Cys552 of FGFR4. We report the cocrystal structure of FGFR4 with CXF-009, which exhibits a dual-warhead covalent binding mode. CXF-009 exhibited stronger selectivity for FGFR4 than FGFR1-3 and other kinases. CXF-009 can also potently inhibit the single cystine mutants, FGFR4(C477A) and FGFR4(C552A), of FGFR4. In summary, our study provides a dual-warhead covalent FGFR4 inhibitor that can covalently target two cysteines of FGFR4. CXF-009, to our knowledge, is the first reported inhibitor that forms dual-warhead covalent bonds with two cysteine residues in FGFR4. CXF-009 also has the potential to overcome drug induced resistant FGFR4 mutations and might serve as a lead compound for future anticancer drug discovery.

Characterization of the cholangiocarcinoma drug pemigatinib against FGFR gatekeeper mutants.

Fibroblast growth factor receptor (FGFR) dysregulation is involved in a variety of tumorigenesis and development. Cholangiocarcinoma is closely related with FGFR aberrations, and pemigatinib is the first drug approved to target FGFR for the treatment of cholangiocarcinoma. Herein, we undertake biochemical and structural analysis on pemigatinib against FGFRs as well as gatekeeper mutations. The results show that pemigatinib is a potent and selective FGFR1-3 inhibitor. The extensive network of hydrogen bonds and van der Waals contacts found in the FGFR1-pemigatinib binding mode accounts for the high potency. Pemigatinib also has excellent potency against the Val-to-Ile gatekeeper mutation but less potency against the Val-to-Met/Phe gatekeeper mutation in FGFR. Taken together, the inhibitory and structural profiles exemplified by pemigatinib may help to thwart Val-to-Ile gatekeeper mutation-based resistance at earlier administration and to advance the further design and improvement for inhibitors toward FGFRs with gatekeeper mutations.

Functional characterization of 27 CYP3A4 protein variants to metabolize regorafenib in vitro.

AIM: Regorafenib is a tyrosine kinase inhibitor that is mainly metabolized by CYP3A4. The genetic polymorphism of CYP3A4 would contribute to differences in metabolism of regorafenib. Previously, we had discovered several novel CYP3A4 variants. However, the catalytic characteristics of these 27 CYP3A4 variants on oxidizing regorafenib have not being determined. The purpose of this study was to investigate the catalytic characteristics of 27 CYP3A4 protein variants on the oxidative metabolism of regorafenib in vitro. METHOD: Wild-type CYP3A4.1 or other variants was incubated with 0.5-20 µmol/L regorafenib for 30 minutes. After sample processing, regorafenib-N-oxide, a primary metabolite, was detected by ultra-performance liquid chromatography-tandem mass spectrometry system. RESULT: CYP3A4.20 had no detectable enzyme activity compared with wild-type CYP3A4.1; five variants (CYP3A4.5, .16, .19, .24, .29) exhibited similar clearance value with CYP3A4.1; four variants (CYP3A4.14, .15, .28, .31) displayed increased enzymatic activities, while remaining variants showed markedly decreased intrinsic clearance values. CONCLUSION: This study is the first to investigate the function of 27 CYP3A4 protein variants on the metabolism of regorafenib in vitro, and it may provide some valuable information for further research in clinic.

Drug-drug interaction study of imatinib and voriconazole in vitro and in vivo.

Background: In clinical practice, common problem polypharmacy could result in the increased risks of drug-drug interactions (DDIs). Co-administered imatinib (IMA) and voriconazole (VOR) as one treatment protocol in cancer patients with fungal infections are common.Purpose: The aim of the present study was to assess the potential DDIs associated with the concurrent use of IMA and VOR in rat liver microsomes (RLMs) and in rats.Methods and results: The concentration levels of IMA, VOR, and their metabolites N-desmethyl IMA (CGP74588) and N-oxide voriconazole (N-oxide VOR) were determined by ultra performance liquid chromatography-tandem mass spectrometry. In vitro study of RLMs, VOR inhibited the IMA metabolism with the half-maximal inhibitory concentration (IC50) of 105.20 µM, while IC50 for IMA against VOR was 61.30 µM. After co-administered IMA and VOR in rats, the C max of IMA was increased significantly, while the AUC0→t, AUC0→∞, and C max of CGP74588 were decreased significantly. In addition, similar results were also found that the main pharmacokinetic parameters (AUC0→t, AUC0→∞, MRT0→∞, T max, and C max) of VOR were increased significantly, while the AUC0→t, AUC0→∞, and C max of N-oxide VOR were decreased significantly. Incorporation of all the results indicated that both drugs had a inhibitory effect on each other's metabolism in vitro and in vivo.Conclusion: Thus, it is of great value to monitor the concomitant use of IMA and VOR in the clinic to reduce the risks of unexpected clinical outcomes.

Inhibitory Mechanisms of Myricetin on Human and Rat Liver Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Myricetin is a flavonoid compound that is abundant in teas, red wine, berries, herbs and vegetables with a variety of pharmacological properties such as antioxidant, anti-inflammatory and anti-cancer effects. Although there are in vitro studies showing that myricetin inhibits human cytochrome P450 (CYP) 2D6 and CYP3A, the inhibitory mechanisms of myricetin on CYP enzymes are still unclear. The aim of this study was to evaluate the inhibitory effects of myricetin on human and rat CYPs, including CYP3A2/3A4, CYP2B1/2B6, CYP2C9/2C11 and CYP2D1/2D6. METHODS: This study was performed to investigate the inhibitory effects of myricetin on human CYP3A4, CYP2B6, CYP2C9, CYP2D6 and rat CYP3A2, CYP2B1, CYP2C11, CYP2D1 through the cocktail approach using ultra-performance liquid chromatography tandem mass spectrometry. Typical probe substrates were used as follows-midazolam for CYP3A2/3A4, dextromethorphan for CYP2D1/2D6, tolbutamide for CYP2C9/2C11, and bupropion for CYP2B1/2B6. RESULTS: The results of this study showed that myricetin might not be a time-dependent inhibitor. Moreover, myricetin inhibited CYP3A4 in an uncompetitive way with an inhibition constant (Ki) value of 143.1 µM. It was also a noncompetitive inhibitor of CYP2C9 and CYP2D6 with Ki values of 31.12 and 53.44 µM and a competitive inhibitor of CYP2B1 with a Ki value of 69.70 µM, as well as a mixed inhibitor of CYP3A2, CYP2C11 and CYP2D1with Ki values of 37.57, 14.88 and 17.39 µM, respectively. CONCLUSIONS: In conclusion, this study indicates that myricetin inhibited CYP3A4/3A2, CYP2C9/2C11, CYP2D6/2D1 and CYP2B1 by various mechanisms with different Ki values. Given that our experiments are established in vitro, further in vivo work is needed to confirm the interaction between myricetin and CYP enzymes, thus providing better guidance for the safe clinical use of myricetin.