The PA28 family proteasome activators play important roles in regulating proteasome activities. Though the three paralogs (PA28α, PA28β, and PA28γ) are similar in terms of primary sequence, they show significant differences in expression pattern, cellular localization and most importantly, biological functions. While PA28αβ is responsible for promoting peptidase activity of proteasome to facilitate MHC-I antigen processing, but unable to promote protein degradation, PA28γ is well-known to not only promote peptidase activity but also proteolytic activity of proteasome. However, why this paralog has the unique function remains elusive. Previous structural studies have mainly focused on mammalian PA28α, PA28β and PA28αβ heptamers, while structural studies on mammalian PA28γ of atomic resolution are still absent to date. In the present work, we determined the Cryo-EM structure of the human PA28γ heptamer at atomic resolution, revealing interesting unique structural features that may hint our understanding the functional mechanisms of this proteasome activator.

1,3-β-Glucan serves as the primary component of the fungal cell wall and is produced by 1,3-β-glucan synthase located in the plasma membrane. This synthase is a molecular target for antifungal drugs such as echinocandins and the triterpenoid ibrexafungerp. In this study, we present the cryo-electron microscopy structure of Saccharomyces cerevisiae 1,3-β-glucan synthase (Fks1) at 2.47-Å resolution. The structure reveals a central catalytic region adopting a cellulose synthase fold with a cytosolic conserved GT-A-type glycosyltransferase domain and a closed transmembrane channel responsible for glucan transportation. Two extracellular disulfide bonds are found to be crucial for Fks1 enzymatic activity. Through structural comparative analysis with cellulose synthases and structure-guided mutagenesis studies, we gain previously unknown insights into the molecular mechanisms of fungal 1,3-β-glucan synthase.

BCR gene fused ABL kinase is the critical driving force for the Philadelphia Chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) and has been extensively explored as a drug target. With a structure-based drug design approach we have discovered a novel inhibitor CHMFL-074, that potently inhibits both the native and a variety of clinically emerged mutants of BCR-ABL kinase. The X-ray crystal structure of CHMFL-074 in complex with ABL1 kinase (PDB ID: 5HU9) revealed a typical type II binding mode (DFG-out) but relatively rare hinge binding. Kinome wide selectivity profiling demonstrated that CHMFL-074 bore a high selectivity (S score(1) = 0.03) and potently inhibited ABL1 kinase (IC50: 24 nM) and PDGFR α/β (IC50: 71 nM and 88 nM). CHMFL-074 displayed strong anti-proliferative efficacy against BCR-ABL-driven CML cell lines such as K562 (GI50: 56 nM), MEG-01 (GI50: 18 nM) and KU812 (GI50: 57 nM). CHMFL-074 arrested cell cycle into the G0/G1 phase and induced apoptosis in the Ph+ CML cell lines. In addition, it potently inhibited the CML patient primary cell's proliferation but did not affect the normal bone marrow cells. In the CML cell K562 inoculated xenograft mouse model, oral administration of 100 mg/kg/d of CHMFL-074 achieved a tumor growth inhibition (TGI) of 65% without exhibiting apparent toxicity. As a potential drug candidate for fighting CML, CHMFL-074 is under extensive preclinical safety evaluation now.

The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.

Treatment of non-small-cell lung cancers (NSCLCs) harboring primary EGFR oncogenic mutations such as L858R and exon 19 deletion delE746_A750 (Del-19) using gefitinib/erlotinib ultimately fails due to the emergence of T790M mutation. Though WZ4002/CO-1686/AZD9291 are effective in overcoming EGFR T790M by targeting Cys797 via covalent bonding, their efficacy is again limited due to the emergence of C797S mutation. New agents effectively inhibiting EGFR T790M without covalent linkage through Cys 797 may solve this problem. We presented here crystal structures of EGFR activating/drug-resistant mutants in complex with a panel of reversible inhibitors along with mutagenesis and enzyme kinetic data. These data revealed a previously un-described hydrophobic clamp structure in the EGFR kinase which may be exploited to facilitate development of next generation drugs targeting EGFR T790M with or without concomitant C797S. Interestingly, mutations in the hydrophobic clamp that hinder drug binding often also weaken ATP binding and/or abolish kinase activity, thus do not readily result in resistance to the drugs.

Non-small-cell lung cancers (NSCLCs) caused by activating mutations in the kinase domain of epidermal growth factor receptor (EGFR) initially respond to first-generation reversible drugs gefitinib and erlotinib. However, clinical efficacy is limited due to the development of drug-resistance that in more than half of the cases are driven by the secondary T790M mutation. CO-1686 is one of the third generation irreversible inhibitors that inhibits EGFR activating mutants, including those with concurrent T790M, while avoiding the off-target toxicity owing to inhibition of wild-type EGFR in treating EGFR mutation-positive NSCLCs. Despite the remarkable success, the experimentally determined structure of this agent in complex with EGFR T790M remains unknown. In this study, we determined crystal structures of EGFR T790M or L858R mutants covalently bound by CO-1686. Based on these structural data, we can explain why CO-1686 irreversibly inhibits EGFR and selectively prefers T790M, which may help improving this or similar compounds, and explain why EGFR L718Q and L844V mutations incur resistance to this agent.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement. However, most patients inevitably develop crizotinib resistance due to acquired secondary mutations in the ALK kinase domain, such as the gatekeeper mutation L1196M and the most refractory mutation, G1202R. Here, we develop XMU-MP-5 as a new-generation ALK inhibitor to overcome crizotinib resistance mutations, including L1196M and G1202R. XMU-MP-5 blocks ALK signaling pathways and inhibits the proliferation of cells harboring either wild-type or mutant EML4-ALK in vitro and suppresses tumor growth in xenograft mouse models in vivo. Structural analysis provides insights into the mode of action of XMU-MP-5. In addition, XMU-MP-5 induces significant regression of lung tumors in two genetically engineered mouse (GEM) models, further demonstrating its pharmacological efficacy and potential for clinical application. These preclinical data support XMU-MP-5 as a novel selective ALK inhibitor with high potency and selectivity. XMU-MP-5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations.

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC50: 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 μM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology.

N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine N(ε)-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.

P53-binding protein 1 (53BP1) plays critical roles in DNA double strand break (DSB) repair by promoting non-homologous end joining (NHEJ), and loss of 53BP1 abolishes PARPi sensitivity in BRCA1-deficient cells by restoring homologous recombination (HR). 53BP1 is one of the proteins initially recruited to sites of DSBs via recognition of H4K20me2 through the Tudor-UDR domain and H2AK15ub through the UDR motif. Although extensive studies have been conducted, it remains unclear how the post-translational modification of 53BP1 affects DSB repair pathway choice. Here, we identified 53BP1 as an acetylated protein and determined that acetylation of 53BP1 inhibit NHEJ and promote HR by negatively regulating 53BP1 recruitment to DSBs. Mechanistically, CBP-mediated acetylation of K1626/1628 in the UDR motif disrupted the interaction between 53BP1 and nucleosomes, subsequently blocking the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs. Hyperacetylation of 53BP1, similar to depletion of 53BP1, restored PARPi resistance in BRCA1-deficient cells. Interestingly, 53BP1 acetylation was tightly regulated by HDAC2 to maintain balance between the HR and NHEJ pathways. Together, our results demonstrate that the acetylation status of 53BP1 plays a key role in its recruitment to DSBs and reveal how specific 53BP1 modification modulates the choice of DNA repair pathway.

Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant BtkC481S poses a major challenge in the management of B-cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib-resistant BtkC481S mutation.

The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M 'gatekeeper' mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib's effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo. These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC.

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct "DFG-in" and "cHelix-out" inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose.

Chitin is one of the most abundant natural biopolymers and serves as a critical structural component of extracellular matrices, including fungal cell walls and insect exoskeletons. As a linear polymer of β-(1,4)-linked N-acetylglucosamine, chitin is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. In this study, we determine seven different cryo-electron microscopy structures of a Saccharomyces cerevisiae chitin synthase in the absence and presence of glycosyl donor, acceptor, product, or peptidyl nucleoside inhibitors. Combined with functional analyses, these structures show how the donor and acceptor substrates bind in the active site, how substrate hydrolysis drives self-priming, how a chitin-conducting transmembrane channel opens, and how peptidyl nucleoside inhibitors inhibit chitin synthase. Our work provides a structural basis for understanding the function and inhibition of chitin synthase.