RESUMO
A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.
Assuntos
Produtos Biológicos/química , Depsipeptídeos/química , Imunoconjugados/química , Substâncias Intercalantes/química , Animais , Antineoplásicos Imunológicos/química , Linhagem Celular Tumoral , DNA/química , Depsipeptídeos/sangue , Depsipeptídeos/farmacocinética , Equinomicina/química , Genes erbB-2 , Meia-Vida , Xenoenxertos , Humanos , Camundongos , Trastuzumab/químicaRESUMO
In this Letter we describe the synthesis and biological evaluation of new benzosuberene analogs with structural modifications on the B-ring. The focus was initially to probe the chemical space around the B-ring C-8 position. This position was readily available for derivatization chemistry using our recently developed new synthesis for this compound class. Furthermore, we describe two new B-ring analogs, one containing a diene and the other a cyclic ether group. Both new analogs show excellent potencies in tumor cell proliferation assays. In addition, we describe molecular modeling studies that provide a binding rationale for reference compound 8 in the colchicine binding site using the known colchicine crystal structure. We also examine whether the cell based potency data obtained with selected new analogs are supported by modeling results.
Assuntos
Derivados de Benzeno/química , Derivados de Benzeno/toxicidade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/toxicidade , Animais , Derivados de Benzeno/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colchicina/análogos & derivados , Cães , Humanos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/metabolismoRESUMO
An experimental approach is described for late-stage lead diversification of frontrunner drug candidates using nanomole-scale amounts of lead compounds for structure-activity relationship development. The process utilizes C-H bond activation methods to explore chemical space by transforming candidates into newly functionalized leads. A key to success is the utilization of microcryoprobe nuclear magnetic resonance (NMR) spectroscopy, which permits the use of low amounts of lead compounds (1-5 µmol). The approach delivers multiple analogues from a single lead at nanomole-scale amounts as DMSO-d6 stock solutions with a known structure and concentration for in vitro pharmacology and absorption, distribution, metabolism, and excretion testing. To demonstrate the feasibility of this approach, we have used the antihistamine agent loratadine (1). Twenty-six analogues of loratadine were isolated and fully characterized by NMR. Informative SAR analogues were identified, which display potent affinity for the human histamine H1 receptor and improved metabolic stability.
Assuntos
Loratadina/análogos & derivados , Loratadina/farmacocinética , Relação Estrutura-Atividade , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dimetil Sulfóxido/química , Cães , Descoberta de Drogas/métodos , Antagonistas não Sedativos dos Receptores H1 da Histamina/química , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacologia , Humanos , Ligação de Hidrogênio , Inativação Metabólica , Loratadina/química , Espectroscopia de Ressonância Magnética , Metaloporfirinas/química , Metaloporfirinas/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
From the beginning of the antibiotic era in the 1940s to the present, Wyeth has sustained an active research program in the area of natural products discovery. This program has continually evolved through the years in order to best align with the "current" drug discovery paradigm in the pharmaceutical industry. The introduction of high-throughput screening and the miniaturization of assays have created a need to optimize natural product samples to better suit these new technologies. Furthermore, natural product programs are faced with an ever shortening time period from hit detection to lead characterization. To address these issues, Wyeth has created a pre-fractionated natural products library using reversed-phase HPLC to complement their existing library of crude extracts. The details of the pre-fractionated library and a cost-benefit analysis will be presented in this review.
Assuntos
Anti-Infecciosos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Bibliotecas de Moléculas Pequenas , Tecnologia Farmacêutica/métodos , Cromatografia Líquida de Alta PressãoRESUMO
The mannopeptimycins (MPPs) are potent glycopeptide antibiotics that contain both D and L forms of the unique, arginine-derived amino acid beta-hydroxyenduracididine (betahEnd). The product of the mppO gene in the MPP biosynthetic cluster resembles several non-heme iron, alpha-ketoglutarate-dependent oxygenases, such as VioC and clavaminate synthase. The role of MppO in betahEnd biosynthesis was confirmed through inactivation of mppO, which yielded a strain that produced dideoxy-MPPs, indicating that mppO is essential for generating the beta-hydroxy functionality for both betahEnd residues. Characterization in vitro of recombinant His6-MppO expressed in E. coli revealed that MppO selectively hydroxylates the beta carbon of free L-enduracididine.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Glicopeptídeos/biossíntese , Imidazolidinas/química , Imidazolidinas/metabolismo , Streptomyces/enzimologia , Glicopeptídeos/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Mutação/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
[structure: see text] Organic extracts of the entomopathogenic fungus Akanthomyces gracilis ARS 2910 contained antibiotics active against Staphylococcus aureus. Bioassay-guided fractionation of the CH2Cl2 extract yielded the antibacterial compound akanthomycin as a mixture of atropisomers along with the closely related compounds 8-methylpyridoxatin and cordypyridone C. Akanthomycin was characterized using X-ray crystallography and NMR.
Assuntos
Antibacterianos/isolamento & purificação , Fungos/química , Piridonas/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Formigas/microbiologia , Cristalografia por Raios X , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Piridonas/isolamento & purificação , Piridonas/farmacologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Animais , Área Sob a Curva , Células Cultivadas , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , Tubulina (Proteína)/metabolismoRESUMO
21-Hydroxyoligomycin A (1) was isolated from Streptomyces cyaneogriseus ssp. noncyanogenus (LL-F28249) and fully characterized by NMR and single-crystal X-ray diffraction methods. The complete 1H and 13C NMR chemical shift assignments for 1 were made using 2D NMR experiments, and the chirality at C-21 was deduced to be R from a J-based configuration analysis. The absolute configuration at C-21 and at the other 18 chiral centers in the molecule were independently confirmed by anomalous dispersion measurements on a crystal of the chloroform methanol solvate of 21-hydroxyoligomycin A (1).
Assuntos
Oligomicinas/química , Streptomyces/química , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , EstereoisomerismoRESUMO
Fermentation extracts of Cordyceps heteropoda (ARSEF #1880), an entomopathogenic fungus isolated from an Australian cicada, yielded a known antifungal compound, myriocin, and a complex microheterogeneous family of novel nonribosomal peptides, each containing two residues of alpha-aminoisobutyric acid (Aib). Structure elucidation of two major components of the peptide mixture, cicadapeptins I and II (1 and 2), was accomplished by amino acid analysis and various MS, 1-D NMR, and 2-D NMR experiments. Both compounds are acylated at the N-terminus by n-decanoic acid and amidated at the C-terminus by 1,2-diamino-4-methylpentane. The amino acid sequence of cicadapeptin I is N-terminus-Hyp-Hyp-Val-Aib-Gln-Aib-Leu-C-terminus. Ile substitutes for Leu in cicadapeptin II. To our knowledge, this is the first report from fungi of consecutive Hyp or Pro residues in a nonribosomal linear peptide. ROESY data indicated that the cicadapeptins adopt a helical conformation. Cicadapeptins I and II displayed antibacterial activity and limited antifungal activity.