RESUMEN
Bioconjugates containing the neuropeptide Y (NPY) analogue [F(7),P(34)]-NPY as targeting moiety are able to deliver toxic agents specifically to breast cancer cells that overexpress the human Y1-receptor (hY1R). To increase their activity, multiple toxophores can be attached to one peptide. Herein, synthesis and characterization of [F(7),P(34)]-NPY conjugates containing two methotrexate (MTX) molecules are presented. First, carboxytetramethylrhodamine was linked to [F(7),P(34)]-NPY by amide or enzymatic linkage. The conjugate containing the enzymatic cleavage site showed high extracellular stability and fast intracellular release. Then, MTX was introduced at positions four and 22 of [F(7),P(34)]-NPY, connected by enzymatic or amide linkage. The toxicity of the analogues on breast cancer cells was hY1R-mediated and dependent on the used linkage and amount of toxophores. Furthermore, conjugates revealed higher potency than MTX on MTX-resistant cells. These results emphasize that peptide-drug conjugates can overcome drug resistance and that the attachment of multiple cleavable toxophores enhances the efficiency of this smart delivery system.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Metotrexato/química , Neuropéptido Y/química , Secuencia de Aminoácidos , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Datos de Secuencia Molecular , Receptores de Neuropéptido Y/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Myxobacterial tubulysins are promising chemotherapeutics inhibiting microtubule polymerization, however, high unspecific toxicity so far prevents their application in therapy. For selective cancer cell targeting, here the coupling of a synthetic cytolysin to the hY1-receptor preferring peptide [F(7),P(34)]-neuropeptide Y (NPY) using a labile disulfide linker is described. Since hY1-receptors are overexpressed in breast tumors and internalize rapidly, this system has high potential as peptide-drug shuttle system. Molecular characterization of the cytolysin-[F(7),P(34)]-NPY bioconjugate revealed potent receptor activation and receptor-selective internalization, while viability studies verified toxicity. Triple SILAC studies comparing free cytolysin with the bioconjugate demonstrated an intracellular mechanism of action regardless of the delivery pathway. Treatments resulted in a regulation of proteins implemented in cell cycle arrest confirming the tubulysin-like effect of the cytolysin. Thus, the cytolysin-peptide bioconjugate fused by a cleavable linker enables a receptor-specific delivery as well as a potent intracellular drug-release with high cytotoxic activity.
Asunto(s)
Sistemas de Liberación de Medicamentos , Neuropéptido Y , Perforina , Receptores de Neuropéptido Y/metabolismo , Animales , Células COS , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Disulfuros/química , Células HEK293 , Humanos , Neuropéptido Y/administración & dosificación , Neuropéptido Y/química , Neuropéptido Y/farmacología , Perforina/administración & dosificación , Perforina/química , Perforina/farmacología , Receptores de Neuropéptido Y/genéticaRESUMEN
The side effects of chemotherapy can be overcome by linking toxic agents to tumor-targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the humanâ Y1 receptor (hY1 R), overexpressed in breast tumors, with analogues of the hY1 R-preferring [F(7) ,P(34) ]NPY. First, carboxytetramethylrhodamine was connected to [F(7) ,P(34) ]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY1 R-mediated delivery and allowed visualization of time-dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F(7) ,P(34) ]NPY by solid-phase peptide synthesis. Internalization studies showed hY1 R subtype selective uptake, and cell viability experiments demonstrated hY1 R-mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore-[F(7) ,P(34) ]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.
Asunto(s)
Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/toxicidad , Neoplasias de la Mama/tratamiento farmacológico , Metotrexato/química , Metotrexato/toxicidad , Neuropéptido Y/química , Neuropéptido Y/metabolismo , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Antimetabolitos Antineoplásicos/efectos adversos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Femenino , Colorantes Fluorescentes , Células HEK293 , Humanos , Metotrexato/efectos adversos , Estructura Molecular , Rodaminas/química , Técnicas de Síntesis en Fase Sólida , Relación Estructura-ActividadRESUMEN
Glycation is a non-enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end-products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE-peptides in enzymatic digests, these species cannot be efficiently detected by LC-ESI-MS-based data-dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGE-peptides and assignment of modification-specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal- and methylglyoxal-derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom-up proteomic approach based on data-dependent acquisition (DDA) LC-MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC-MS/MS (DDA). Altogether 44 hydroimidazolone- and dihydroxyimidazolidine-derived peptides representing 42 AGE-modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients.
Asunto(s)
Arginina/análisis , Arginina/química , Productos Finales de Glicación Avanzada/análisis , Fragmentos de Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Arginina/sangre , Arginina/metabolismo , Diabetes Mellitus/sangre , Productos Finales de Glicación Avanzada/sangre , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Tripsina/metabolismoRESUMEN
Most toxic agents currently used for chemotherapy show a narrow therapeutic window, because of their inability to distinguish between healthy and cancer cells. Targeted drug delivery offers the possibility to overcome this issue by selectively addressing structures on the surface of cancer cells, therefore reducing undesired side effects. In this broad field, peptide-drug conjugates linked by intracellular cleavable structures have evolved as highly promising agents. They can specifically deliver toxophores to tumor cells by targeting distinct receptors overexpressed in cancer. In this review, we focus on these compounds and describe important factors to develop a highly efficient peptide-drug conjugate. The necessary properties of tumor-targeting peptides are described, and the different options for cleavable linkers used to connect toxic agents and peptides are discussed, and synthetic considerations for the introduction of these structures are reported. Furthermore, recent examples and current developments of peptide-drug conjugates are critically evaluated with a special focus on the applied linker structures and their future use in cancer therapy.
Asunto(s)
Preparaciones de Acción Retardada/síntesis química , Portadores de Fármacos/síntesis química , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citotoxinas/química , Citotoxinas/metabolismo , Citotoxinas/uso terapéutico , Preparaciones de Acción Retardada/metabolismo , Portadores de Fármacos/metabolismo , Diseño de Fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Relación Estructura-ActividadRESUMEN
Diabetes-induced hyperglycemia increases the extracellular concentration of methylglyoxal. Methylglyoxal-derived hydroimidazolones (MG-H) form advanced glycation end products (AGEs) that accumulate in the serum of diabetic patients. The binding of hydroimidozolones to the receptor for AGEs (RAGE) results in long-term complications of diabetes typified by vascular and neuronal injury. Here we show that binding of methylglyoxal-modified albumin to RAGE results in signal transduction. Chemically synthesized peptides containing hydroimidozolones bind specifically to the V domain of RAGE with nanomolar affinity. The solution structure of an MG-H1-V domain complex revealed that the hydroimidazolone moiety forms multiple contacts with a positively charged surface on the V domain. The high affinity and specificity of hydroimidozolones binding to the V domain of RAGE suggest that they are the primary AGE structures that give rise to AGEs-RAGE pathologies.