In silico strategies for the rational design, synthesis, and biological evaluation of ligands targeting macromolecules of pharmaceutical interest

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes, widely expressed in all kingdoms of life and encoded by eight evolutionarily unrelated gene families: α-, β-, γ-, δ-, ζ-, η-, θ- and ι-CAs. The CAs catalyze the reversible hydration of carbon dioxide into bicarbonate and proton that is physiologically crucial for all living beings because involved in respiration, pH and CO2 homeostasis, transport of CO2/HCO3- and a multitude of biosynthetic reactions. In Homo sapiens, fifteen α-class CA isoforms were identified, which are implicated in a plethora of physiological processes such as electrolytes secretion in many tissues/organs, metabolic reactions (e.g. gluconeogenesis, lipogenesis, ureagenesis), bone resorption, calcification, and carcinogenesis. Thus, an abnormal expression/activity of specific human CAs results in a multitude of human pathological processes that can be targeted with a pharmacological intervention based on CA modulation. Moreover, numerous α-, β-, γ- and η-CAs were identified in many bacteria, protozoa, and fungi that act as human pathogens. In this context, CAs were shown to be crucial for the virulence, growth or acclimatization of the parasites in the hosts. Their inhibition produces growth impairment and defects in the pathogen, being a promising strategy for chemotherapy. The research activity included in this Ph.D. thesis fits in the context of the spreading interest of the scientific community on CAs as drug targets for the treatment of a multitude of disorders. Thus, a set of projects involving drug-design, synthesis, biological evaluation and in silico investigation of the ligand-target interactions of new CA inhibitors (CAIs) were the focus of the three-year Ph.D. cycle. In the first reported project, the concept of tail- (and dual tail) approach was extended up to the three tail approach. This study aimed to increase the selectivity of the benzenesulfonamide scaffold against certain CA isoforms over others, through the introduction of three pendants called “tails” with different lipophilic/hydrophilic nature. Hence, it was possible both to target simultaneously the most variable residues of the inner/outer rim of the hydrophobic and hydrophilic half of the active site and to interact with the peculiar accessory subpockets which differentiate the various isoforms. The synthesized three-tailed inhibitors (TTIs) resulted to be more selective against the glaucoma-associated CA isoforms (hCA II, IV and XII) with respect to the mono-tailed precursors. A massive in silico study of the TTIs-targets interactions was carried out to extend the X-ray crystallography results and the IOP-lowering ability of the three most promising derivatives was evaluated. The second project adopted the multi-targets approach for the treatment of multifactorial diseases, such as inflammation and tumors. Inhibitors of target CA isoforms, to repristinate the correct synovial fluid pH value (against IV, IX and XII), were endowed with an H2S releasing ability, to exploit the gasotransmitter relieving effect in inflammation, developing innovative H2S releaser-CAI hybrids. The stopped-flow kinetic assay pointed out selective inhibition profiles against CAs IX and XII for most synthesized compounds over the off-target CAs I and II. Four compounds were submitted to the Paw pressure and Incapacitance tests to evaluate their ability as anti-inflammatory agents. In the third project, an in silico study was conducted to optimize the CA IV inhibitory properties of a set of benzenesulfonamide derivatives, that produced potent but unselective CA IV inhibition. A peculiar hydrophilic cleft of the CA IV active site (pocket A) was the target for the drug optimization process. The introduction of a pendant with an H-bond acceptor/donor group in a position suitable for the interaction with pocket A was predicted to improve the binding to the target CA and not to off-target isoforms. As a result, the newly reported set of derivatives showed enhanced CA inhibition up to a subnanomolar range. The research of new CAI chemotypes is an important strategy to selectively inhibit specific isoforms over others. In the fourth and fifth projects, the joint use of docking studies, MM-GBSA and molecular dynamic (MD) simulations allowed to evaluate the binding mode of derivatives with CAI scaffolds other than sulfonamides such as hydantoin, saccharin and acesulfame. A zinc binder inhibition mechanism was computed for the hydantoins, which are able to exist in a deprotonated form at the physiological pH. A mechanism based on the anchorage to zinc-bound water molecule was instead proposed and assessed for tertiary sulfonamides, saccharin- and acesulfame derivatives. The sixth project was here reported as representative for a series of in silico investigations carried out to figure out the SAR of several sets of benzenesulfonamide CAIs. The design of N-nitrosulfonamide silver salts was reported to combine the compounds selective inhibition of certain pathogens CAs (over human isozymes) and the antiseptic properties of silver. Indeed, a subset of such compounds showed effective and selective inhibition of the CAs from Trypanosoma cruzi (TcCA) and Leishmania donovani chagasi (LdcCA) compared to hCAs I and II as well as effective chemotherapic action in vitro against several protozoan strains and forms. Another project consisted of the in silico characterization of the binding mode of benzoxaborole derivatives within the α-, β-, and γ-CA active site of the bacterial Vibrio cholerae (VchCA). Primarily the targets homology models were built to proceed therefore with docking studies, MM-GBSA and MD calculations with the boron-based ligands. The computational analysis, for the first time carried out on a γ-class CA, showed that benzoxaborole derivatives adopt both a tetrahedral and trigonal bipyramidal zinc coordination in complex with VchCA (α-CA), a tetrahedral geometry within VchCAβ and two alternative penta-coordinated geometries around the zinc ion in VchCAγ active site. GABAA receptors (GABAARs) are another crucial target for the treatment of several human diseases, such as anxiety disorders, insomnia, epilepsy, restlessness, and aggressive behaviors. Many clinically important drugs were discovered to target GABAARs, interacting at different allosteric binding sites such as benzodiazepine and benzodiazepine site ligands, barbiturates, intravenous and volatile anesthetics, anticonvulsants, ethanol and neuroactive steroids, and performing their pharmacological effects. Nowadays, several natural products such as flavonoids, menthol, magnolol, honokiol, coronaridines and others, were found to modulate GABAARs. All of these compounds exhibit diverse pharmacology mediated by known and unknown binding sites and their potential for clinical application is currently being explored. In this scenario, the identification of the binding site and the investigation of the binding mode of coronaridine congeners, such as (+)-catharanthine, is an actual challenge. The joint use of electrophysiological, radioligand displacement and in silico studies promoted the identification of the (+)-catharanthine binding site at the β(+)α(-), concluding that this ligand acts according to a loreclezole-like mechanism. Finally, during my stage in the organic chemistry laboratory of Prof. Vittorio Pace (University of Vienna) I was involved in a study on the reductive lithiation arene-catalyzed of imines as a new method for the synthesis of amino alcohols.