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1.
J Struct Biol ; 211(1): 107512, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32325220

RESUMO

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.


Assuntos
Dipeptidases/ultraestrutura , Epitopos/ultraestrutura , Imunoconjugados/ultraestrutura , Anticorpos/química , Anticorpos/imunologia , Anticorpos/ultraestrutura , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Proteínas de Membrana/imunologia , Proteínas de Membrana/ultraestrutura , Proteólise
2.
Bioorg Med Chem Lett ; 28(3): 437-440, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29287958

RESUMO

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.


Assuntos
Acrilamidas/farmacologia , Citocinas/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Acrilamidas/síntese química , Acrilamidas/química , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade
3.
Biochem Biophys Res Commun ; 491(3): 681-686, 2017 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-28756225

RESUMO

Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD+), and NAD+ depletion ultimately results in cell death. The rate limiting step within the NAD+ salvage pathway required for converting nicotinamide to NAD+ is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD+ depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD+ synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD+ de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic.


Assuntos
Cianetos/administração & dosagem , Citocinas/antagonistas & inibidores , Citocinas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/metabolismo , Guanidinas/administração & dosagem , NAD/biossíntese , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Anilidas , Apoptose/efeitos dos fármacos , Apoptose/genética , Arginina/análogos & derivados , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Fibrossarcoma/genética , Humanos , Mutação/genética , NAD/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resultado do Tratamento
4.
Bioorg Med Chem Lett ; 27(15): 3317-3325, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28610984

RESUMO

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Citocinas/química , Citocinas/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoindóis/química , Isoindóis/farmacocinética , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Relação Estrutura-Atividade , Ureia/farmacocinética , Ureia/uso terapêutico
5.
Biopolymers ; 105(4): 234-43, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26756394

RESUMO

Unlike other known anti-fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (KD ∼ 70 pM) and a fast association rate (kon ∼ 2 × 10(7) M(-1 ) s(-1) ). The three-dimensional structure of the Fab 43.1-fluorescein complex was resolved at 2.4 Å resolution. The antibody binding site is exclusively assembled by the CDR loops. It is comprised of a 14 Å groove-shaped entrance leading to a 9 Å by 7 Å binding pocket. The highly polar binding pocket complementary encloses the fluorescein's carboxyphenyl moiety and tightly fixes it by multiple hydrogen bonds. The fluorescein's xanthene ring is embedded in the more hydrophobic groove and stacked between the side chains of Tyr37L and of Arg99H providing conditions for an excited state electron transfer process. In comparison to fluorescein, the absorption spectrum of the complex in the visible region is shifted to the "red" by 23 nm. The complex demonstrates a very weak fluorescence (Φc = 0.0018) with two short lifetime components: 0.03 ns (47%) and 0.8 ns (24%), which reflects a 99.8% fluorescein emission quenching effect upon complex formation. The antibody 43.1 binds fluorescein with remarkable affinity, fast association rate, and strongly quenches its emission. Therefore, it may present a practical interest in applications such as molecular sensors and switches.


Assuntos
Anticorpos Monoclonais/química , Fluoresceína/química , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Cinética , Conformação Proteica , Termodinâmica
6.
Biochemistry ; 53(23): 3719-26, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24832237

RESUMO

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 107 M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻5 s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.


Assuntos
Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Antagonistas do Ácido Fólico/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Metotrexato/antagonistas & inibidores , Modelos Moleculares , Água/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Cristalografia por Raios X , Antagonistas do Ácido Fólico/metabolismo , Hibridomas , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Imunossupressores/antagonistas & inibidores , Imunossupressores/química , Imunossupressores/metabolismo , Ligantes , Metotrexato/química , Metotrexato/metabolismo , Camundongos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica , Água/química
7.
Sci Transl Med ; 16(739): eadd8936, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38507467

RESUMO

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.


Assuntos
Anticorpos , Artrite Experimental , Imunoconjugados , Esteroides , Humanos , Animais , Camundongos , Preparações Farmacêuticas , Receptores de Glucocorticoides/uso terapêutico , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Modelos Animais de Doenças , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico
8.
Bioorg Med Chem Lett ; 23(12): 3487-90, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23664214
9.
Artigo em Inglês | MEDLINE | ID: mdl-22691797

RESUMO

Successfully forming ligand-protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand- and detergent-exchange method was developed to obtain ligand-protein complexes of an adamantane series of compounds with 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures.


Assuntos
Cristalografia por Raios X/métodos , Desenho de Fármacos , Inibidores Enzimáticos/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Cristalografia por Raios X/instrumentação , Humanos , Ligantes , Modelos Moleculares , Estrutura Quaternária de Proteína
10.
ACS Chem Biol ; 17(3): 556-566, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35188729

RESUMO

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.


Assuntos
Proteínas Serina-Treonina Quinases , Linfócitos T , Fatores Imunológicos , Imunoterapia , Transdução de Sinais
11.
Sci Rep ; 11(1): 566, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436675

RESUMO

Melanotransferrin (MTf) is an iron-binding member of the transferrin superfamily that can be membrane-anchored or secreted in serum. On cells, it can mediate transferrin-independent iron uptake and promote proliferation. In serum, it is a transcytotic iron transporter across the blood-brain barrier. MTf has been exploited as a drug delivery carrier to the brain and as an antibody-drug conjugate (ADC) target due to its oncogenic role in melanoma and its elevated expression in triple-negative breast cancer (TNBC). For treatment of TNBC, an MTf-targeting ADC completed a phase I clinical trial (NCT03316794). The structure of its murine, unconjugated Fab fragment (SC57.32) is revealed here in complex with MTf. The MTf N-lobe is in an active and iron-bound, closed conformation while the C-lobe is in an open conformation incompatible with iron binding. This combination of active and inactive domains displays a novel inter-domain arrangement in which the C2 subdomain angles away from the N-lobe. The C2 subdomain also contains the SC57.32 glyco-epitope, which comprises ten protein residues and two N-acetylglucosamines. Our report reveals novel features of MTf and provides a point of reference for MTf-targeting, structure-guided drug design.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Domínios Proteicos , Acetilglucosamina , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Expressão Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/fisiologia , Ferro/metabolismo , Macaca fascicularis , Melanoma/etiologia , Melanoma/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Neoplasias de Mama Triplo Negativas/genética
12.
ACS Med Chem Lett ; 12(7): 1108-1115, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34267880

RESUMO

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

13.
J Med Chem ; 64(1): 417-429, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33378180

RESUMO

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.


Assuntos
Produtos Biológicos/síntese química , Desenho de Fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Regulação Alostérica , Animais , Artrite Reumatoide/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Ligantes , Camundongos , Fator de Necrose Tumoral alfa/metabolismo
14.
Proteins ; 76(3): 536-47, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19274732

RESUMO

B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Cristalografia por Raios X , Peptídeo Natriurético Encefálico/química , Animais , Linhagem Celular , Epitopos/química , Camundongos , Conformação Proteica , Termodinâmica
15.
J Med Chem ; 61(3): 1153-1163, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29342358

RESUMO

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species. Replacement of the dihydrouracil in 1 with an N-linked uracil provided better potency in the genotype 1 replicon assay. Results from phase 1 clinical studies supported once-daily oral dosing with ABT-072 in HCV infected patients. A phase 2 clinical study that combined ABT-072 with the HCV protease inhibitor ABT-450 provided a sustained virologic response at 24 weeks after dosing (SVR24) in 10 of 11 patients who received treatment.


Assuntos
Citosina/análogos & derivados , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Estilbenos/química , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Disponibilidade Biológica , Técnicas de Química Sintética , Citosina/síntese química , Citosina/química , Citosina/farmacocinética , Citosina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Permeabilidade , Estereoisomerismo , Sulfonamidas/química , Sulfonamidas/farmacocinética , Distribuição Tecidual , Proteínas não Estruturais Virais/química
16.
Mol Cancer Ther ; 16(7): 1236-1245, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28468779

RESUMO

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Citocinas/genética , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Med Chem ; 49(21): 6416-20, 2006 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17034148

RESUMO

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.


Assuntos
Inibidores de Adenosina Desaminase , Inibidores da Dipeptidil Peptidase IV , Glicoproteínas/antagonistas & inibidores , Hipoglicemiantes/síntese química , Piridinas/síntese química , Pirrolidinas/síntese química , Adenosina Desaminase/química , Administração Oral , Animais , Sítios de Ligação , Células CACO-2 , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/química , Cães , Feminino , Intolerância à Glucose/tratamento farmacológico , Glicoproteínas/química , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Macaca fascicularis , Modelos Moleculares , Estrutura Molecular , Piridinas/farmacocinética , Piridinas/farmacologia , Pirrolidinas/farmacocinética , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Estereoisomerismo , Relação Estrutura-Atividade
18.
Protein Sci ; 14(12): 3039-47, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16322581

RESUMO

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.


Assuntos
Inibidores Enzimáticos/química , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/química , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeo Sintases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência , Especificidade por Substrato
19.
Bioorg Med Chem Lett ; 16(24): 6226-30, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17010607

RESUMO

A series of xanthine mimetics containing 5,5 and 5,6 heterocycle fused imidazoles were synthesized as dipeptidyl peptidase IV inhibitors. Compound 7 is potent (h-DPPIV K(i)=2nM) and exhibits excellent selectivity and no species specificity against rat and human enzymes. The X-ray structure confirms that the binding mode of 7 to rat DPPIV is similar to the parent xanthines.


Assuntos
Inibidores da Dipeptidil Peptidase IV , Inibidores de Proteases/farmacologia , Xantinas/farmacologia , Animais , Dipeptidil Peptidase 4/química , Imidazóis/farmacologia , Cinética , Modelos Moleculares , Inibidores de Proteases/síntese química , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Difração de Raios X , Xantinas/síntese química
20.
Chem Biol Drug Des ; 67(1): 58-65, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16492149

RESUMO

The D-Ala-D-Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency. We report here a multidisciplinary approach to this effort that allowed for rapid generation of cocrystal structures, thereby providing the crystallographic information critical for driving the inhibitor optimization process. This effort resulted in the discovery of low-nanomolar inhibitors of this bacterial enzyme.


Assuntos
Inibidores Enzimáticos/química , Peptídeo Sintases/antagonistas & inibidores , Relação Estrutura-Atividade , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Concentração Inibidora 50 , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Especificidade por Substrato , Sulfonamidas/química , Sulfonamidas/metabolismo
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