RESUMO
Techniques facilitating the synthesis and screening of very high diversity nonstandard macrocyclic peptide libraries have led to such compounds receiving increasing attention as potential drug candidates. Specifically, approaches which allow the use of non-proteinogenic amino acids are proving to be particularly effective, since they expand the accessible chemical space of the starting library and thus allow the identification of compounds with structural similarity to known drugs. This review focuses on mRNA display screening platforms for drug discovery and their combined use with genetic code reprogramming to identify novel macrocyclic peptides with high affinities for disease-related targets of interest.
Assuntos
Peptídeos/genética , Ligantes , Compostos Macrocíclicos , RNA Mensageiro/genéticaRESUMO
We designed and synthesized a tandem-hairpin motif of pyrrole (P)-imidazole (I) polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates (1) that targets the human telomere repeat sequence 5'-d(CCCTAA)n-3'. As a control, conjugate 2 (hairpin PI polyamide with seco-CBI), which also targets the human telomere repeat sequence, was synthesized. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) using 5' Texas Red-labeled 219-bp DNA fragments revealed the outstandingly high sequence selectivity of 1, with no mismatch alkylation. Furthermore, an evaluation performed in human cancer cell lines demonstrated that conjugate 1 has low cytotoxicity compared with conjugate 2. In addition, a cell-staining analysis indicated that conjugate 1 induced apoptosis moderately by DNA damage. This study demonstrated that conjugate 1 can be used as an effective alkylator for telomere repeat sequences or as an apoptotic inducer.
Assuntos
Alquilantes/farmacologia , Antineoplásicos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Pirróis/farmacologia , Telômero/efeitos dos fármacos , Sequências Repetidas Terminais/efeitos dos fármacos , Alquilantes/síntese química , Alquilantes/química , Alquilação , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Poliacrilamida , Humanos , Imidazóis/química , Indóis/química , Conformação Molecular , Pirróis/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Hairpin N-methylpyrrole-N-methylimidazole polyamide seco-CBI conjugates 2-6 were designed for synthesis by Fmoc solid-phase synthesis, and their DNA-alkylating activities against the Kras codonâ 13 mutation were compared by high-resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codonâ 13 mutation site, with alkylation occurring at the A of the sequence 5'-ACGTCACCA-3' (siteâ 2), including minor 1â bp-mismatch alkylation against wild type 5'-ACGCCACCA-3' (siteâ 3). Conjugate 6, which differs from conjugate 5 by exchanging one Py unit with a ß unit, showed high selectivity but only weakly alkylated the A of 5'-ACGTCACCA-3' (siteâ 2). The hairpin polyamide seco-CBI conjugate 5 thus alkylates according to Dervan's pairing rule with the pairing recognition which ß/ß pair targets T-A and A-T pairs. SPR and a computer-minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.
Assuntos
DNA/química , Imidazóis/química , Nylons/química , Pirróis/química , Proteínas ras/genética , Alanina/genética , Alanina/metabolismo , Alquilação , Pareamento de Bases , Sequência de Bases , Códon , DNA/metabolismo , Humanos , Modelos Moleculares , Mutação , Nylons/síntese química , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Imidazóis/síntese química , Imidazóis/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Nylons/síntese química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HT29 , Humanos , Imidazóis/farmacologia , Camundongos Nus , Mutação , Nylons/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Tandem N-methylpyrrole-N-methylimidazole (Py-Im) polyamides with good sequence-specific DNA-alkylating activities have been designed and synthesized. Three alkylating tandem Py-Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10â bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high-resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1, which contained a ß-alanine linker, displayed the most-selective sequence-specific alkylation towards the target 10â bp DNA sequence. The tandem Py-Im polyamide conjugates displayed greater sequence-specific DNA alkylation than conventional hairpin Py-Im polyamide conjugates (4 and 5). For further research, the design of tandem Py-Im polyamide conjugates could play an important role in targeting specific gene sequences.