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 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.

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.

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 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.

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.

Targeting MCL-1 in cancer: current status and perspectives.

Myeloid leukemia 1 (MCL-1) is an antiapoptotic protein of the BCL-2 family that prevents apoptosis by binding to the pro-apoptotic BCL-2 proteins. Overexpression of MCL-1 is frequently observed in many tumor types and is closely associated with tumorigenesis, poor prognosis and drug resistance. The central role of MCL-1 in regulating the mitochondrial apoptotic pathway makes it an attractive target for cancer therapy. Significant progress has been made with regard to MCL-1 inhibitors, some of which have entered clinical trials. Here, we discuss the mechanism by which MCL-1 regulates cancer cell apoptosis and review the progress related to MCL-1 small molecule inhibitors and their role in cancer therapy.

Mechanism of forkhead transcription factors binding to a novel palindromic DNA site.

Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors.

Structural basis for DNA recognition by FOXC2.

The FOXC family of transcription factors (FOXC1 and FOXC2) plays essential roles in the regulation of embryonic, ocular, and cardiac development. Mutations and abnormal expression of FOXC proteins are implicated in genetic diseases as well as cancer. In this study, we determined two crystal structures of the DNA-binding domain (DBD) of human FOXC2 protein, in complex with different DNA sites. The FOXC2-DBD adopts the winged-helix fold with helix H3 contributing to all the base specific contacts, while the N-terminus, wing 1, and the C-terminus of FOXC2-DBD all make additional contacts with the phosphate groups of DNA. Our structural, biochemical, and bioinformatics analyses allow us to revise the previously proposed DNA recognition mechanism and provide a model of DNA binding for the FOXC proteins. In addition, our structural analysis and accompanying biochemical assays provide a molecular basis for understanding disease-causing mutations in FOXC1 and FOXC2.

Structural basis of a novel heterodimeric Fc for bispecific antibody production.

Bispecific antibodies provide an efficient tool for combinational clinical therapy. Here we have engineered a heterodimeric Fc for bispecific antibodies production by combining the knob-into-hole and electrostatic steering strategies where a bulky hydrophobic residue Phe405 of the IgG CH3 interface is mutated to a charged residue Lys and Lys409 of the corresponding CH3 domain is mutated to Ala. The crystal structure of this Fc heterodimer solved here at 2.7Å resolution revealed how these two mutations resulted a complementary binding interface and explained why F405K mutation could effectively inhibit Fc homodimer formation during protein expression. An anti-HER2 bispecific antibody derived from trastuzumab and pertuzumab was generated by this heterodimeric Fc. It showed comparable or improved efficacy than the combination of trastuzumab and pertuzumab in inhibiting proliferation of cancer cells in vitro and in vivo. Overall this study shows that the heterodimeric Fc engineered here provides an efficient platform for generating active bispecific antibody for cancer treatment.

Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade.

The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy.