Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction toward CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy toward the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed Ki of 0.49 and 4.7 μM toward CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1. [Figure not available: see fulltext.].

Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...

Made available in DSpace on 2016-08-17T18:39:36Z (GMT). No. of bitstreams: 1 3549.pdf: 14954889 bytes, checksum: bc938e517ca5db95a3d7b52d18aea9a8 (MD5) Previous issue date: 2011-03-24 / Financiadora de Estudos e Projetos / Compounds of the pirrolbenzodiazepine (PBD) family are known for their promising antitumor activity. Among these, the hybrids, those that have a portion PDB a chain spacer and another functional group, such as the coumarins of this work, have been extensively explored. It is also known that these compounds bind to DNA, but there is no structural data showing how it occurs. To overcome this lack of information molecular docking calculations were performed to study the formation of complexes between these PBD-hybrids and DNA. The compounds were modeled and the coordinates of complexes DNA-receptors with different ligands were obtained from the Protein Data Bank. The redocking served to validate the conditions of the experiments and the scores were used as the parameter to evaluate the complexes formed. The analysis of the intermolecular interactions, an essential knowledge for understanding the obtained structures were analyzed using high-resolution molecular imaging. The results of the in silico experiments showed the formation of complexes in the mixed-mode with the PBD ligand moiety intercalating between the DNA bases and the coumarin portion occupying the minor groove, and a preference for intercalation between GG bases. Moreover, it is possible to postulate that the complex becomes an adduct with the formation of a covalent bond between the intercalated portion PBD and a nucleotide base G. Finally, a correlation between the docking results and the biological activities of the studied compounds was established. / Compostos da família das pirrolbenzodiazepinas (PBD) são conhecidos por apresentarem atividade antitumoral promissora. Dentre elas, as chamadas híbridas que possuem uma porção PDB uma cadeia espaçadora e outro grupo funcional, como as cumarinas deste trabalho, têm sido muito exploradas. Sabe-se que estes compostos se ligam ao DNA, mas não há dados estruturais mostrando como a ligação ocorre. Para suprir esta falta de informação foram realizados cálculos de docking molecular para estudar a formação de complexos entre estas PBDs híbridas e o DNA. Os compostos estudados foram modelados e as coordenadas de complexos DNA-receptores com diferentes ligantes foram obtidas do Protein Data Bank. O redocking serviu para validar as condições dos experimentos e os escores foram utilizados como parâmetro de avaliação dos complexos formados. A análise das interações intermoleculares, conhecimento essencial para o entendimento das estruturas obtidas, foi feita utilizando visualização molecular de alta resolução. Os resultados dos experimentos in silico mostraram a formação de complexos no modo de ligação misto, com os ligantes intercalando a porção PBD entre bases do DNA e a porção cumarina ocupando o sulco menor, mostrando ter preferência pela intercalação entre bases GG. Mais ainda, é possível postular que o complexo se torne um aduto com a formação de uma ligação covalente entre a porção PBD intercalada e uma base nucleotídica G. Finalmente foi estabelecida uma correlação entre os resultados do docking e as atividades biológicas dos compostos estudados.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...

The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 μM concentration. Further concentration-response analysis revealed two compounds, and , as potent and selective CB2 ligands with sub-micromolar activities ( = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound were sought. Compound was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.