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1.
J Pharm Biomed Anal ; 210: 114566, 2022 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-35042144

RESUMEN

Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.


Asunto(s)
Diabetes Mellitus Tipo 2 , Péptido 1 Similar al Glucagón , Albúminas , Animales , Semivida , Hipoglucemiantes , Liraglutida , Péptidos , Ratas
2.
J Org Chem ; 85(3): 1466-1475, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-31660743

RESUMEN

The Myc transcription factor represents an "undruggable" target of high biological interest due to its central role in various cancers. An abbreviated form of the c-Myc protein, called Omomyc, consists of the Myc DNA-binding domain and a coiled-coil region to facilitate dimerization of the 90 amino acid polypeptide. Here we present our results to evaluate the synthesis of Omomyc using three complementary strategies: linear Fmoc solid-phase peptide synthesis (SPPS) using several advancements for difficult sequences, native chemical ligation from smaller peptide fragments, and a high-throughput bacterial expression and assay platform for rapid mutagenesis. This multifaceted approach allowed access to up to gram quantities of the mini-protein and permitted in vitro and in vivo SAR exploration of this modality. DNA-binding results and cellular activity confirm that Omomyc and analogues presented here, are potent binders of the E-box DNA engaged by Myc for transcriptional activation and that this 90-amino acid mini-protein is cell permeable and can inhibit proliferation of Myc-dependent cell lines. We also present additional results on covalent homodimerization through disulfide formation of the full-length mini-protein and show the coiled-coil region can be truncated while preserving both DNA binding and cellular activity. Altogether, our results highlight the ability of advanced peptide synthesis to achieve SAR tractability in a challenging synthetic modality.


Asunto(s)
ADN , Proteínas Proto-Oncogénicas c-myc , Línea Celular , ADN/metabolismo , Fragmentos de Péptidos , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo
3.
Mol Cell Biol ; 39(22)2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31501275

RESUMEN

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Genes myc , Fragmentos de Péptidos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/farmacología , Proteínas Recombinantes/farmacología , Transcripción Genética , Activación Transcripcional
4.
Sci Rep ; 8(1): 585, 2018 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-29330364

RESUMEN

A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells. After two or three rounds of selection, 7.5% of randomly picked clones were GLP1R/GCGR co-agonists, and a further 1.53% were agonists of a single receptor. The phages were sequenced and 35 corresponding peptides were synthesized. 18 peptides were potent co-agonists, 8 of whom showed EC50 ≤ 30 pM on each receptor, comparable to the best rationally designed co-agonists reported in the literature. Based on literature examples, two sequences were engineered to stabilize against dipeptidyl peptidase IV cleavage and prolong the in vivo half-life: the engineered peptides were comparably potent to the parent peptides on both receptors, highlighting the potential use of phage-derived peptides as therapeutic agents. The strategy described here appears of general value for the discovery of optimized polypharmacology paradigms across several metabolically-related hormones.


Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos/síntesis química , Péptidos/farmacología , Receptores de Glucagón/agonistas , Diabetes Mellitus/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Humanos , Obesidad/tratamiento farmacológico , Biblioteca de Péptidos , Péptidos/genética , Polifarmacia , Análisis de Secuencia de ADN
6.
Bioorg Med Chem Lett ; 19(15): 4042-5, 2009 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-19553107

RESUMEN

PARP inhibitors have been demonstrated to retard intracellular DNA repair and therefore sensitize tumor cells to cytotoxic agents or ionizing radiation. We report the identification of a novel class of PARP1 inhibitors, containing a pyrrolo moiety fused to a dihydroisoquinolinone, derived from virtual screening of the proprietary collection. SAR exploration around the nitrogen of the aminoethyl appendage chain of 1 led to compounds that displayed low nanomolar activity in a PARP1 enzymatic assay.


Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Quinolonas/química , Antineoplásicos/farmacología , Sitios de Unión , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Reparación del ADN , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Ligandos , Modelos Químicos , Polímeros/química , Relación Estructura-Actividad
7.
J Med Chem ; 50(20): 4953-75, 2007 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-17824681

RESUMEN

The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.


Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , Integrasa de VIH/química , VIH-1/efectos de los fármacos , Morfolinas/síntesis química , Pirimidinonas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Perros , Inhibidores de Integrasa VIH/farmacocinética , Inhibidores de Integrasa VIH/farmacología , VIH-1/enzimología , VIH-1/fisiología , Humanos , Macaca mulatta , Morfolinas/farmacocinética , Morfolinas/farmacología , Unión Proteica , Pirimidinonas/farmacocinética , Pirimidinonas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
8.
J Med Chem ; 50(9): 2225-39, 2007 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-17428043

RESUMEN

Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC95 of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.


Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , VIH-1/efectos de los fármacos , Piridinas/síntesis química , Pirimidinas/síntesis química , Animales , Disponibilidad Biológica , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Perros , Inhibidores de Integrasa VIH/farmacocinética , Inhibidores de Integrasa VIH/farmacología , Semivida , Humanos , Macaca mulatta , Unión Proteica , Piridinas/química , Piridinas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Relación Estructura-Actividad , Replicación Viral
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