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.

Structural characterization of the DNA binding mechanism of retinoic acid-related orphan receptor gamma.

Retinoic acid-related orphan receptor gamma (RORγ) plays critical roles in regulating various biological processes and has been linked to immunodeficiency disorders and cancers. DNA recognition is essential for RORγ to exert its functions. However, the underlying mechanism of the DNA binding by RORγ remains unclear. In this study, we present the crystal structure of the complex of RORγ1 DNA-binding domain (RORγ1-DBD)/direct repeat DNA element DR2 at 2.3 Å resolution. We demonstrate that RORγ1-DBD binds the DR2 motif as a homodimer, with the C-terminal extension (CTE) region of RORγ1-DBD contributing to the DNA recognition and the formation of dimeric interface. Further studies reveal that REV-ERB-DBD and RXR-DBD, also bind the DR2 site as a homodimer, while NR4A2-DBD binds DR2 as a monomer. Our research uncovers a binding mechanism of RORγ1 to the DR2 site and provides insights into the biological functions of RORγ1 and the broader RORs subfamily.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

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.

Structural insight into the macrocyclic inhibitor TPX-0022 of c-Met and c-Src.

c-Met has been an attractive target of prognostic and therapeutic studies in various cancers. TPX-0022 is a macrocyclic inhibitor of c-Met, c-Src and CSF1R kinases and is currently in phase I/II clinical trials in patients with advanced solid tumors harboring MET gene alterations. In this study, we determined the co-crystal structures of the c-Met/TPX-0022 and c-Src/TPX-0022 complexes to help elucidate the binding mechanism. TPX-0022 binds to the ATP pocket of c-Met and c-Src in a local minimum energy conformation and is stabilized by hydrophobic and hydrogen bond interactions. In addition, TPX-0022 exhibited potent activity against the resistance-relevant c-Met L1195F mutant and moderate activity against the c-Met G1163R, F1200I and Y1230H mutants but weak activity against the c-Met D1228N and Y1230C mutants. Overall, our study reveals the structural mechanism underlying the potency and selectivity of TPX-0022 and the ability to overcome acquire resistance mutations and provides insight into the development of selective c-Met macrocyclic inhibitors.

Structural basis of the specificity and interaction mechanism of Bmf binding to pro-survival Bcl-2 family proteins.

The apoptotic pathway is regulated by protein-protein interactions between members of the Bcl-2 family. Pro-survival Bcl-2 family proteins act as cell guardians and protect cells against death. Selective binding and neutralization of BH3-only proteins with pro-survival Bcl-2 family proteins is critical for initiating apoptosis. In this study, the binding assay shows that the BH3 peptide derived from the BH3-only protein Bmf has a high affinity for the pro-survival proteins Bcl-2 and Bcl-xL, but a much lower affinity for Mcl-1. The complex structures of Bmf BH3 with Bcl-2, Bcl-xL and Mcl-1 reveal that the α-helical Bmf BH3 accommodates into the canonical groove of these pro-survival proteins, but the conformational changes and some interactions are different among the three complexes. Bmf BH3 forms conserved hydrophobic and salt bridge interactions with Bcl-2 and Bcl-xL, and also establishes several hydrogen bonds to support their binding. However, the highly conserved Asp-Arg salt bridge is not formed in the Mcl-1/Bmf BH3 complex, and few hydrogen bonds are observed. Furthermore, mutational analysis shows that substitutions of less-conserved residues in the α2-α3 region of these pro-survival Bcl-2 family proteins, as well as the highly conserved Arg, lead to significant changes in their binding affinity to Bmf BH3, while substitutions of less-conserved residues in Bmf BH3 have a more dramatic effect on its affinity to Mcl-1. This study provides structural insight into the specificity and interaction mechanism of Bmf BH3 binding to pro-survival Bcl-2 family proteins, and helps guide the design of BH3 mimics targeting pro-survival Bcl-2 family proteins.

Structures of p53/BCL-2 complex suggest a mechanism for p53 to antagonize BCL-2 activity.

Mitochondrial apoptosis is strictly controlled by BCL-2 family proteins through a subtle network of protein interactions. The tumor suppressor protein p53 triggers transcription-independent apoptosis through direct interactions with BCL-2 family proteins, but the molecular mechanism is not well understood. In this study, we present three crystal structures of p53-DBD in complex with the anti-apoptotic protein BCL-2 at resolutions of 2.3-2.7 Å. The structures show that two loops of p53-DBD penetrate directly into the BH3-binding pocket of BCL-2. Structure-based mutations at the interface impair the p53/BCL-2 interaction. Specifically, the binding sites for p53 and the pro-apoptotic protein Bax in the BCL-2 pocket are mostly identical. In addition, formation of the p53/BCL-2 complex is negatively correlated with the formation of BCL-2 complexes with pro-apoptotic BCL-2 family members. Defects in the p53/BCL-2 interaction attenuate p53-mediated cell apoptosis. Overall, our study provides a structural basis for the interaction between p53 and BCL-2, and suggests a molecular mechanism by which p53 regulates transcription-independent apoptosis by antagonizing the interaction of BCL-2 with pro-apoptotic BCL-2 family members.

Structural basis of the farnesoid X receptor/retinoid X receptor heterodimer on inverted repeat DNA.

Farnesoid X receptor (FXR) is a ligand-activated transcription factor known as bile acid receptor (BAR). FXR plays critical roles in various biological processes, including metabolism, immune inflammation, liver regeneration and liver carcinogenesis. FXR forms a heterodimer with the retinoid X receptor (RXR) and binds to diverse FXR response elements (FXREs) to exert its various biological functions. However, the mechanism by which the FXR/RXR heterodimer binds the DNA elements remains unclear. In this study, we aimed to use structural, biochemical and bioinformatics analyses to study the mechanism of FXR binding to the typical FXRE, such as the IR1 site, and the heterodimer interactions in the FXR-DBD/RXR-DBD complex. Further biochemical assays showed that RAR, THR and NR4A2 do not form heterodimers with RXR when bound to the IR1 sites, which indicates that IR1 may be a unique binding site for the FXR/RXR heterodimer. Our studies may provide a further understanding of the dimerization specificity of nuclear receptors.

Targeting p53 pathways: mechanisms, structures, and advances in therapy.

The TP53 tumor suppressor is the most frequently altered gene in human cancers, and has been a major focus of oncology research. The p53 protein is a transcription factor that can activate the expression of multiple target genes and plays critical roles in regulating cell cycle, apoptosis, and genomic stability, and is widely regarded as the "guardian of the genome". Accumulating evidence has shown that p53 also regulates cell metabolism, ferroptosis, tumor microenvironment, autophagy and so on, all of which contribute to tumor suppression. Mutations in TP53 not only impair its tumor suppressor function, but also confer oncogenic properties to p53 mutants. Since p53 is mutated and inactivated in most malignant tumors, it has been a very attractive target for developing new anti-cancer drugs. However, until recently, p53 was considered an "undruggable" target and little progress has been made with p53-targeted therapies. Here, we provide a systematic review of the diverse molecular mechanisms of the p53 signaling pathway and how TP53 mutations impact tumor progression. We also discuss key structural features of the p53 protein and its inactivation by oncogenic mutations. In addition, we review the efforts that have been made in p53-targeted therapies, and discuss the challenges that have been encountered in clinical development.

Structural insight into the ligand binding mechanism of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors, plays important roles in regulating xenobiotic metabolism, cellular differentiation, stem cell maintenance, as well as immunity. More recently, AHR has gained significant interest as a drug target for the development of novel cancer immunotherapy drugs. Detailed understanding of AHR-ligand binding has been hampered for decades by the lack of a three-dimensional structure of the AHR PAS-B domain. Here, we present multiple crystal structures of the Drosophila AHR PAS-B domain, including its apo, ligand-bound, and AHR nuclear translocator (ARNT) PAS-B-bound forms. Together with biochemical and cellular assays, our data reveal structural features of the AHR PAS-B domain, provide insights into the mechanism of AHR ligand binding, and provide the structural basis for the future development of AHR-targeted therapeutics.

Corrigendum to: "Farnesoid X receptor (FXR): Structures and ligands" [Comput. Struct. Biotechnol. J. 19 (2021) 2148-2159].

[This corrects the article DOI: 10.1016/j.csbj.2021.04.029.].

Characterization of the modification of Kelch-like ECH-associated protein 1 by different fumarates.

Fumarates (fumaric acid esters), primarily dimethyl fumarate (DMF) and monoethyl fumarate (MEF) and its salts, are orally administered systemic agents used for the treatment of psoriasis and multiple sclerosis. It is widely believed that the pharmaceutical activities of fumarates are exerted through the Keap1-Nrf2 pathway. Although it has been revealed that DMF and MEF differentially modify specific Keap1 cysteine residues and result in the differential activation of Nrf2, how the modification of DMF and MEF impacts the biochemical properties of Keap1 has not been well characterized. Here, we found that both DMF and MEF can only modify the BTB domain of Keap1 and that only C151 is accessible for covalent binding in vitro. Dynamic fluorescence scanning (DSF) assays showed that the modification of DMF to Keap1 BTB increased its thermal stability, while the modification of MEF dramatically decreased its thermal stability. Further crystal structures revealed no significant conformational variation between the DMF-modified and MEF-modified BTBs. Overall, our biochemical and structural study provides a better understanding of the covalent modification of fumarates to Keap1 and may suggest fundamentally different mechanisms adopted by fumarates in regulating the Keap1-Nrf2 pathway.

Structural study of ponatinib in inhibiting SRC kinase.

Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.

Structural insight into the molecular mechanism of cilofexor binding to the farnesoid X receptor.

Farnesoid X receptor (FXR) is a bile acid-related nuclear receptor and is considered a promising target to treat several liver disorders. Cilofexor is a selective FXR agonist and has already entered phase III trials in primary sclerosing cholangitis (PSC) patients. Pruritis caused by cilofexor treatment is dose dependent. The binding characteristics of cilofexor with FXR and its pruritogenic mechanism remain unclear. In our research, the affinity of cilofexor bound to FXR was detected using an isothermal titration calorimetry (ITC) assay. The binding mechanism between cilofexor and FXR-LBD is explained by the cocrystal structure of the FXR/cilofexor complex. Structural models indicate the possibility that cilofexor activates Mas-related G protein-coupled receptor X4 (MRGPRX4) or G protein-coupled bile acid receptor 1 (GPBAR1), leading to pruritus. In summary, our analyses provide a molecular mechanism of cilofexor binding to FXR and provide a possible explanation for the dose-dependent pruritis of cilofexor.

Structural insights into the potency and selectivity of covalent pan-FGFR inhibitors.

FIIN-2, TAS-120 (Futibatinib) and PRN1371 are highly potent pan-FGFR covalent inhibitors targeting the p-loop cysteine of FGFR proteins, of which TAS-120 and PRN1371 are currently in clinical trials. It is critical to analyze their target selectivity and their abilities to overcome gatekeeper mutations. In this study, we demonstrate that FIIN-2 and TAS-120 form covalent adducts with SRC, while PRN1371 does not. FIIN-2 and TAS-120 inhibit SRC and YES activities, while PRN1371 does not. Moreover, FIIN-2, TAS-120 and PRN1371 exhibit different potencies against different FGFR gatekeeper mutants. In addition, the co-crystal structures of SRC/FIIN-2, SRC/TAS-120 and FGFR4/PRN1371 complexes reveal structural basis for kinase targeting and gatekeeper mutations. Taken together, our study not only provides insight into the potency and selectivity of covalent pan-FGFR inhibitors, but also sheds light on the development of next-generation FGFR covalent inhibitors with high potency, high selectivity, and stronger ability to overcome gatekeeper mutations.

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.

Farnesoid X receptor (FXR): Structures and ligands.

Farnesoid X receptor (FXR) is a bile acid activated nuclear receptor (BAR) and is mainly expressed in the liver and intestine. Upon ligand binding, FXR regulates key genes involved in the metabolic process of bile acid synthesis, transport and reabsorption and is also involved in the metabolism of carbohydrates and lipids. Because of its important functions, FXR is considered as a promising drug target for the therapy of bile acid-related liver diseases. With the approval of obeticholic acid (OCA) as the first small molecule to target FXR, many other small molecules are being evaluated in clinical trials. This review summarizes the structures of FXR, especially its ligand binding domain, and the development of small molecules (including agonists and antagonists) targeting FXR.

Structural insight into the molecular mechanism of p53-mediated mitochondrial apoptosis.

The tumor suppressor p53 is mutated in approximately half of all human cancers. p53 can induce apoptosis through mitochondrial membrane permeabilization by interacting with and antagonizing the anti-apoptotic proteins BCL-xL and BCL-2. However, the mechanisms by which p53 induces mitochondrial apoptosis remain elusive. Here, we report a 2.5 Å crystal structure of human p53/BCL-xL complex. In this structure, two p53 molecules interact as a homodimer, and bind one BCL-xL molecule to form a ternary complex with a 2:1 stoichiometry. Mutations at the p53 dimer interface or p53/BCL-xL interface disrupt p53/BCL-xL interaction and p53-mediated apoptosis. Overall, our current findings of the bona fide structure of p53/BCL-xL complex reveal the molecular basis of the interaction between p53 and BCL-xL, and provide insight into p53-mediated mitochondrial apoptosis.