Carbonic anhydrases (CAs, E.C. 220.127.116.11.) are ubiquitous metalloenzymes present throughout most organisms in all kingdom of life and they are classified in eight evolutionary unrelated gene families namely α-, β-, γ-, δ-, ε-, ζ-, η-, θ-, ι-CAs. CAs catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. In many organisms, these enzymes are involved in CO2 and pH homeostasis, transport of CO2/HCO3-, respiration and a multitude of biosynthetic reaction. In humans, fifteen isoenzymes belonging to α-class have been identified (hCAs I-IV, VA and VB, VI-XIV). Since many hCAs have been implicated and extensively studied in a multitude of physiological processes (gluconeogenesis, lipogenesis, bone resorption, calcification, tumorigenesis, etc.), the abnormal activity or deregulated expression of specific hCAs have been linked with a wide array of pathological consequences (glaucoma, epilepsy, obesity, cancer, etc.). For these reasons, in the recent years several hCAs (VII, IX and XII) became well-established targets for designing CAs modulators endowed with therapeutic and/or diagnostic applications. Although a large number of molecules have been designed, synthesized and tested for their affinity against hCAs, their use as drugs is still limited due to the lack of selectivity for a specific isoform. Hence, the main challenge in the development of new CA inhibitors is to find selectivity against isoforms involved in human diseases and to prevent off-target related side effects that are caused by the inhibition of the ubiquitous isoforms hCA I and II. In this scenario, the basis for the rational drug design of more selective compounds can be provided by structural data on hCA isoforms collected so far and by the inspection of their complexes with inhibitor molecules. A careful comparison between all the crystallized hCAs revealed that the main differences in terms of oligomeric structure and sequence among the isoenzymes, are observed in the region comprising residues 127-136, which thus has to be considered the main suggestion in the drug designing of novel selective hCAs inhibitors. For example, residue 131, located within this “selective pocket” is not conserved among the different isoforms and it has been well explained that it plays a critical role in determining compound selectivity (Fig.1). In general, these structural differences allow the compounds to easily enter the active site and bind in a conformation that favors interactions with residues in the hydrophobic pocket controlling selectivity. This “selective valley” located in the rim of the active site could be targeted to find isoform specific inhibitors. The most popular drug design strategy employed to strike this specific area is the so called “tail approach”. This consists in the design of a molecular template containing the portion addressed to catalytic cavity through the zinc-binder group and an additional fragment (namely “tail”) to the core structure molecule for enhancing interactions with specific hydrophobic or hydrophilic residues lined the middle area or the rim of the active site. In the last decade Gitto R. and coworkers at University of Messina contributed to expand the chemical space of compounds which affect the catalytic activity of CAs, reporting a collection of classical CAIs bearing sulfonamide moiety. Among them, several inhibitors proved to be active in the low nanomolar range, but most of them were not characterized by a good selectivity profile against the druggable isoforms hCA VII, IX and XII. Nonetheless, the discovery of these CAIs and the resolution of the X-ray complexes for the most active compounds, highlighted new suggestions for the development of novel series of human CAIs that could specifically bind the target isoforms. The first part of the research activity of this PhD project focused on the development of new class of compounds strictly related to previously discovered sulfonamides. The main goal was the improvement of our knowledge about structure-affinity relationships (SARs). Thus, we planned the synthesis of novel sulfonamide-based CAIs bearing azepine/piperidine/piperazine core that differ mainly in the “tail” molecules. This wide set of cicloalkylamine derivatives were tested against selected hCAs isoforms. Then, structural and computational studies were carried out in order to investigate the binding pose of the most intriguing compounds and to identify residues within hCAs active site involved in the stabilization of the protein-ligand complexes. As second project of this PhD period, novel series of non-classical CAIs have been investigated. Based on the evidence that some coumarins showed high affinity and selectivity against the tumor associated hCA IX and XII, the natural compound Umbelliferon (7-hydroxycoumarin) was chosen as template for our novel design. By inserting different substituents on coumarin core, we obtained a small library of coumarin derivatives. Thus, the evaluation of inhibitory activity revealed that these compounds possessed nanomolar affinity and high selectivity toward tumor-associated hCA IX and XII. As it was recently found, three classes of CAs that are α-, β-, γ-CAs, seem to play a crucial role in the growth, virulence and acclimation for several pathogenic bacteria in the host organism. As consequence, prokaryotic CAs represent excellent drug targets for the development of novel chemotherapic agents. Based on this assumption, in the last part of this PhD course, we discovered potent and selective inhibitors against the α, β and γ classes of the pathogenic species Vibrio Cholerae (VchCAα, β, γ) by means of computer assisted drug design. Notably, we disclosed the first modelled “open active site” of VchCAβ, as useful tool for the investigation of binding mode of VchCAs inhibitors.
|Titolo:||Targeting Carbonic Anhydrases (CAs): Rational Design, Synthesis, Structural Studies and Biochemical Evaluation|
|Data di pubblicazione:||8-gen-2021|
|Appare nelle tipologie:||Tesi di dottorato|