Computational approaches in drug discovery: strategies to address polypharmacology and the synthesis/optimization of lead compounds

This Ph.D. thesis focused on the application of molecular modeling methods to design and optimize compounds with affinity and selectivity against therapeutic targets of interest, such as carbonic anhydrases (CAs), G-quadruplexes (G4s), β3-adrenergic receptors (β3-ARs), and DNA damage inducible 1 homolog 2 (DDI2). 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 catalyse 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. The initial phase of the work is dedicated to the application of techniques and methodologies aimed at the selective inhibition of specific CA isoforms. In the first reported project, the concept of three tail approach and ring approach were investigated. This study aimed to increase the potency and selectivity of the 1,3,4-thiadiazole-2-sulfonamide scaffold against proper hCAs, 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 middle/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 cancer-associated CA isoforms (hCA IX, IV and XII) with respect to the ubiquitous CA (I and II). The second project adopted the ring approach for the development of selective hCA XIII inhibitors as new oral male contraceptives. The selective inhibition of the target isoform, exploiting the peculiar V200 of the target in the inner rim of the active site, could exert a profound influence on the processes of sperm development, maturation and capacitation, making these compounds promising candidates for the reduction of sperm fertility. The stopped-flow kinetic assay pointed out selective inhibition profiles against CAs XIII for all synthesized compounds over the off-target CAs I and II. In the third project, an extensive in silico investigation and enzyme kinetics studies were conducted to evaluate the inhibitory activity of various compounds targeting tumor-associated CA IX and XII with the aim to identify a relationship between SAR and binding modes. Moreover, numerous α-, β-, γ-, and ι-CAs were identified in many bacteria 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. Therefore the fourth project focused on bacterial CAs, with the objective of developing innovative antibiotic agents to combat drug-resistant pathogens. This investigation included the evaluation of compounds against specific isoforms in Mycobacterium tuberculosis (MtCA1, MtCA2, and MtCA3) and Vibrio cholerae (VchCAα, VchCAβ, and VchCAγ). In the absence of structural data for some isoforms, in silico studies employed the construction of homology models, thus aiding in the identification of promising candidates with specific activity profiles against these bacterial targets. The use of polypharmacology, which involves the simultaneous administration of multiple drugs, can enhance therapeutic response through synergistic action on multiple targets. This approach is widely employed in the treatment of multifactorial diseases, such as cancer, and also in the antibiotic field to prevent the onset of drug resistance. The development of dual-target molecular hybrids, where two distinct pharmacological entities are combined into a single chemical entity, retains the benefits of polypharmacology while minimizing its disadvantages, such as drug-drug interactions and reduced patient compliance. In this scenario the following project comprised the design and synthesis of dual-target compounds, characterised by both carbonic anhydrase inhibitory (CAI) and a penicillin-binding protein (PBP) targeting moiety, with the objective of enhancing antibacterial efficacy and reduce the onset of drug-resistance. Subsequently, these hybrid compounds were evaluated in silico by covalent docking studies to predict their interaction profile with the PBP of Neisseria gonorrhoeae. All the compounds were submitted to stopped-flow kinetic assays in different bacterial CAs, and the best compounds were evaluated against different strains of clinical and multidrug-resistant N. gonorrhoeae. The fifth project also involves the development of dual-target compounds, exhibiting the capacity to inhibit CAs and to stabilise non-canonical DNA structures (specifically G-quadruplex), with the objective of achieving a synergistic anti-cancer effect. This required the acquisition of structural data through the utilization of X-ray crystallography and mass spectrometry, of previously developed berberine derivatives and a new acridine derivative. The obtained information allowed for the future development of new compounds with dual activity. In the context of anticancer research, the sixth project was oriented towards the development of selective antagonists of the β3-ARs as novel anti-cancer agents. β3-ARs play a complex role in cancer biology, influencing tumor growth, metastasis, and the tumor microenvironment. These receptors, primarily known for their role in adipose tissue and metabolism, have been implicated in promoting cancer cell survival and proliferation through adrenergic signalling pathways. Activation of β3-ARs can lead to increased angiogenesis, immune modulation, and resistance to apoptosis, making them potential targets for cancer therapy. Blocking β3-ARs in certain cancers has shown promise in reducing tumor progression and may improve the effectiveness of existing treatments. In order to obtain selective β3-ARs blockers, an in silico design process was employed which involved virtual screening of a comprehensive compound library, combined with docking studies of known β3-antagonists. This process led to the development of three new series of compounds. Several of these compounds exhibited significant potency and selectivity toward the target receptor, highlighting their potential as effective therapeutic candidates. Finally, in the appendix, following a collaboration request from Prof. Giada Bianchi of Harvard Medical School, Boston, USA, new inhibitors of human DNA damage inducible 1 homolog 2 (DDI2) were designed in silico and synthesized.