RESUMEN
Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full structural characterisation. Among these, the first thiophene derivative of reblastatin showed strong antiproliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed, revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of Hsps is the target site for the new geldanamycin derivatives.
Asunto(s)
Antineoplásicos/farmacología , Benzoquinonas/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Benzoquinonas/química , Benzoquinonas/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de Choque Térmico/metabolismo , Helicobacter pylori/química , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/aislamiento & purificación , Estructura Molecular , Streptomyces/química , Relación Estructura-ActividadRESUMEN
Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence-labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell-based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP-binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Quinonas/farmacología , Streptomyces/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Proteínas de Choque Térmico/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinonas/síntesis química , Quinonas/química , Streptomyces/química , Streptomyces/genética , Relación Estructura-ActividadRESUMEN
Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/química , Streptomyces/metabolismo , Antineoplásicos/metabolismo , Calorimetría/métodos , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fluorobencenos/metabolismo , Fluorobencenos/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Modelos Moleculares , Quinonas/química , Streptomyces/genética , metaminobenzoatos/metabolismo , metaminobenzoatos/farmacologíaRESUMEN
Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 µM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas Inmovilizadas/antagonistas & inhibidores , Análisis por Matrices de Proteínas/métodos , Benzoquinonas/química , Benzoquinonas/metabolismo , Benzoquinonas/farmacología , Unión Competitiva , Relación Dosis-Respuesta a Droga , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Lactamas Macrocíclicas/farmacología , Estabilidad Proteica , Reproducibilidad de los ResultadosRESUMEN
Covering 2005 to 2013. In this review recent progress in the development of heat shock proteins (Hsp90) in oncogenesis is illuminated. Particular emphasis is put on inhibitors such as geldanamycin and analogues that serve as a natural product show case. Hsp90 has emerged as an important target in cancer therapy and/or against pathogenic cells which elicit abnormal Hsp patterns. Competition for ATP by geldanamycin and related compounds abrogate the chaperone function of Hsp90. In this context, this account pursues three topics in detail: a) Hsp90 and its biochemistry, b) Hsp90 and its role in oncogenesis and c) strategies to create compound libraries of structurally complex inhibitors like geldanamycin on which SAR studies and the development of drugs that are currently in different stages of clinical testing rely.
Asunto(s)
Antineoplásicos , Benzoquinonas , Productos Biológicos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Benzoquinonas/síntesis química , Benzoquinonas/química , Benzoquinonas/uso terapéutico , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/uso terapéutico , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/uso terapéutico , Estructura MolecularRESUMEN
Supplementing a culture of a mutant strain of Actinosynnema pretiosum that is unable to biosynthesize aminohydroxy benzoic acid (AHBA), with 3-azido-5-hydroxy-benzoic acid and 3-azido-5-amino-benzoic acid, unexpectedly yielded anilino ansamitocins instead of the expected azido derivatives. This is the first example of the bioreduction of organic azides. The unique nature of these results was demonstrated when 3-azido-5-amino-benzoic acid was fed to the corresponding AHBA blocked mutant of Streptomyces hygroscopicus, the geldanamycin producer. This mutasynthetic experiment yielded the fully processed azido derivative of geldanamycin.
Asunto(s)
Aminobenzoatos/farmacología , Antibacterianos/síntesis química , Azidas/química , Benzoquinonas/síntesis química , Hidroxibenzoatos/farmacología , Lactamas Macrocíclicas/síntesis química , Maitansina/análogos & derivados , Streptomyces/química , Aminobenzoatos/química , Antibacterianos/química , Antibacterianos/farmacología , Azidas/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Hidroxibenzoatos/química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Maitansina/síntesis química , Maitansina/química , Maitansina/farmacología , Estructura Molecular , Streptomyces/genética , Streptomyces/metabolismoAsunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Maitansina/análogos & derivados , Actinomycetales/genética , Actinomycetales/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Benzoquinonas/síntesis química , Benzoquinonas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/metabolismo , Maitansina/síntesis química , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Streptomyces/genética , Streptomyces/metabolismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.