RESUMO
Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp3 carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Enabled by a combination of smart library design and machine learning, we engineer the iron/α-ketoglutarate dependent halogenase WelO5* for the late-stage functionalization of the complex and chemically difficult to derivatize macrolides soraphen A and C, potent anti-fungal agents. While the wild type enzyme WelO5* does not accept the macrolide substrates, our engineering strategy leads to active halogenase variants and improves upon their apparent kcat and total turnover number by more than 90-fold and 300-fold, respectively. Notably, our machine-learning guided engineering approach is capable of predicting more active variants and allows us to switch the regio-selectivity of the halogenases facilitating the targeted analysis of the derivatized macrolides' structure-function activity in biological assays.
Assuntos
Algoritmos , Macrolídeos/metabolismo , Oxirredutases/metabolismo , Engenharia de Proteínas , Biocatálise , Biotransformação , Fungos/fisiologia , Halogenação , Macrolídeos/química , Modelos Moleculares , Oxirredutases/químicaRESUMO
The glidobactin-like natural products (GLNPs) glidobactinâ A and cepafunginâ I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Assuntos
Photorhabdus/genética , Inibidores de Proteassoma/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Escherichia coli/metabolismo , Família Multigênica/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Photorhabdus/metabolismo , Inibidores de Proteassoma/química , Relação Estrutura-AtividadeRESUMO
The peptidic ß-lactone proteasome inhibitors (PIs) cystargolides A and B were used to conduct structure-activity relationship (SAR) studies in order to assess their anticancer potential. A total of 24 different analogs were designed, synthesized and evaluated for proteasome inhibition, for cytotoxicity towards several cancer cell lines, and for their ability to enter intact cells. X-ray crystallographic analysis and subunit selectivity was used to determine the specific subunit binding associated with the structural modification of the ß-lactone (P1), peptidic core, (Px and Py), and end-cap (Pz) of our scaffold. The cystargolide derivative 5k, structurally unique at both Py and P1, exhibited the most promising inhibitory activity for the ß5 subunit of human proteasomes (IC50â¯=â¯3.1â¯nM) and significant cytotoxicity towards MCF-7 (IC50â¯=â¯416â¯nM), MDA-MB-231 (IC50â¯=â¯74â¯nM) and RPMI 8226 (IC50â¯=â¯41â¯nM) cancer cell lines. Cellular infiltration assays revealed that minor structural modifications have significant effects on the ability of our PIs to inhibit intracellular proteasomes, and we identified 5k as a promising candidate for continued therapeutic studies. Our novel drug lead 5k is a more potent proteasome inhibitor than carfilzomib with mid-to-low nanomolar IC50 measurements and it is cytotoxic against multiple cancer cell lines at levels approaching those of carfilzomib.
Assuntos
Antineoplásicos/farmacologia , Dipeptídeos/farmacologia , Desenho de Fármacos , Lactonas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Dipeptídeos/química , Relação Dose-Resposta a Droga , Humanos , Lactonas/síntese química , Lactonas/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteassoma/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Herein is described a convergent enantioselective route to an advanced intermediate in the synthesis of schiglautone A, a Schisandra triterpenoid with an unusual architecture. The synthetic route to this intermediate displaying 6 of the 7 stereocenters builds upon two fragments, an aldehyde elaborated from the Wieland-Miescher ketone, and a ketone. The preparation of the latter features a lithiation-borylation enzymatic resolution sequence, which led to the formation of the desired product with high enantio- and diastereoselectivities. After aldol coupling of the two fragments, the final quaternary stereocenter was installed by cyclopropane opening. The functionalized intermediate was isolated as a single diastereoisomer and thus offers a valuable starting point for further synthetic exploration.
Assuntos
Triterpenos/síntese química , Estrutura Molecular , Estereoisomerismo , Triterpenos/químicaRESUMO
Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the ß5i substrate-binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.
Assuntos
Fatores Imunológicos/química , Modelos Moleculares , Oligopeptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/química , Humanos , Estrutura Molecular , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
One life, two strategies: Crucial structural differences between the human and the Plasmodium falciparum proteasomes were recently identified. A combination of cryo-EM and functional characterization enabled the design of a selective antimalarial proteasome inhibitor that shows low toxicity in the host. When used with artemisinin, this ligand offers a new approach for the efficient treatment of malaria at all stages of the parasite lifecycle.
Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Testes de Sensibilidade Parasitária , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/químicaRESUMO
The epipolythiodioxopiperazine (ETP) gliotoxin mediates toxicity via its reactive thiol groups and thereby contributes to virulence of the human pathogenic fungus Aspergillus fumigatus. Self-intoxication of the mold is prevented either by reversible oxidation of reduced gliotoxin or by irreversible conversion to bis(methylthio)gliotoxin. The latter is produced by the S-methyltransferase TmtA and attenuates ETP biosynthesis. Here, we report the crystal structure of TmtA in complex with S-(5'-adenosyl)-l-homocysteine. TmtA features one substrate and one cofactor binding pocket per protein, and thus, bis-thiomethylation of gliotoxin occurs sequentially. Molecular docking of substrates and products into the active site of TmtA reveals that gliotoxin forms specific interactions with the protein surroundings, and free energy calculations indicate that methylation of the C10a-SH group precedes alkylation of the C3-SH site. Altogether, TmtA is well suited to selectively convert gliotoxin and to control its biosynthesis, suggesting that homologous enzymes serve to regulate the production of their toxic natural sulfur compounds in a similar manner.
Assuntos
Gliotoxina/antagonistas & inibidores , Metiltransferases/metabolismo , Humanos , Metiltransferases/química , Modelos MolecularesRESUMO
This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This suggests that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.