RESUMO
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Assuntos
Regulação Alostérica , Descoberta de Drogas , Inibidores Enzimáticos , Proteômica , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Masculino , Camundongos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Cisteína/efeitos dos fármacos , Cisteína/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Instabilidade de Microssatélites , Modelos Moleculares , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Morte Celular/efeitos dos fármacos , Trifosfato de Adenosina/metabolismoRESUMO
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.
Assuntos
Desenho de Fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Terapia de Alvo Molecular/métodos , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/uso terapêutico , Toxinas Botulínicas/classificação , Toxinas Botulínicas/metabolismo , Simulação por Computador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Temperatura Alta , Humanos , Influenza Humana/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Estabilidade Proteica , Proteínas/imunologia , Proteínas/metabolismo , TemperaturaRESUMO
Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.
Assuntos
Flavinas/metabolismo , NADP/metabolismo , Prótons , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Catálise , Domínio Catalítico , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.
RESUMO
CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP , Neoplasias Hepáticas , Masculino , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , LigantesRESUMO
The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.
Assuntos
Epitopos/metabolismo , Galactose/análogos & derivados , Glicoproteínas/metabolismo , Lampreias/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Fucose/metabolismo , Galactose/metabolismo , Glicoproteínas/química , Glicosilação , Células HEK293 , Humanos , Lampreias/imunologia , Ligantes , Espectrometria de Massas/métodos , Ácido N-Acetilneuramínico/metabolismo , Sulfatos/metabolismo , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.
Assuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Evolução Molecular Direcionada/métodos , Anticorpos Anti-HIV/imunologia , Imunogenicidade da Vacina , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/química , Regiões Determinantes de Complementaridade/imunologia , Modelos Animais de Doenças , Células HEK293 , Anticorpos Anti-HIV/química , Humanos , Camundongos , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologiaRESUMO
Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate ß-hydroxyl or ß-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite ß-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.
Assuntos
Metiltransferases/química , Policetídeo Sintases/química , Policetídeos/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Cianobactérias/enzimologia , Metilação , Metiltransferases/genética , Mutagênese Sítio-Dirigida , Mutação , Myxococcales/enzimologia , Policetídeo Sintases/genética , Conformação Proteica , Domínios Proteicos , Especificidade por SubstratoRESUMO
Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camelídeos Americanos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/química , Anticorpos Antivirais/ultraestrutura , Cristalografia por Raios X , Cães , Feminino , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Domínio ÚnicoRESUMO
Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Engenharia de Proteínas/métodos , Multimerização Proteica , Sítios de Ligação , Ligação ProteicaRESUMO
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.
Assuntos
Metiltransferases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Metiltransferases/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.
Assuntos
Materiais Biomiméticos/química , Sondas Moleculares/química , Policetídeo Sintases/química , Policetídeos/química , Streptomyces/química , Sequência de Aminoácidos , Materiais Biomiméticos/síntese química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Sondas Moleculares/síntese química , Dados de Sequência Molecular , NADP/química , Policetídeo Sintases/genética , Policetídeos/síntese química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Cinética , Nucleotídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products and is catalyzed by multiple families of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (MTs). Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent MT. Crystal structures were determined for MycE bound to the product S-adenosyl-L-homocysteine (AdoHcy) and magnesium, both with and without the natural substrate mycinamicin VI. This represents the first structure of a natural product sugar MT in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic MT domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel MT organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active-site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order-disorder transition in the active site relative to the S-adenosyl-L-homocysteine complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.