The search for new compounds with a given biological activity requires enormous effort in terms of manpower and cost. This effort arises from the large number of compounds that need to be synthesized and subsequently biologically evaluated. For this reason the pharmaceutical industry has shown great interest in theoretical methods that enable the rational design of pharmaceutical agents. In the last years bioinformatics has experienced a great evolution due to the development of specialized software and to the increasing computer power. The codification of the structural information of molecules through molecular descriptors and the subsequent data analysis allow establishing QSAR models (Quantitative Structure-Activity Relationship) that can be applied to the design and the virtual screening of new drugs. The development of sophisticated Docking methodologies also allows a more accurate predict of the biological activity of molecules. Moreover, through this type of computational techniques and theoretical approaches, it is possible to develop explanatory hypothesis on the mechanism of action of drugs. This work provides a brief description of a series of studies implemented in the software MOE (Molecular Operating Environment) with particular attention to the medicinal chemistry aspects.

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.

Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.

5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer, is representative of a new class of CK2 inhibitors with K(i) values in the low nanomolar range and unprecedented selectivity versus other kinases. Here we present the crystal structure of the complexes of CX-4945 and two analogues (CX-5011 and CX-5279) with the catalytic subunit of human CK2. Consistent with their ATP-competitive mode of inhibition, all three compounds bind in the active site of CK2 (type I inhibitors). The tricyclic scaffold of the inhibitors superposes on the adenine of ATP, establishing multiple hydrophobic interactions with the binding cavity. The more extended scaffold, as compared to that of ATP, allows the carboxylic function, shared by all three ligands, to penetrate into the deepest part of the active site where it makes interactions with conserved water W1 and Lys-68, thus accounting for the crucial role of this negatively charged group in conferring high potency to this class of inhibitors. The presence of a pyrimidine in CX-5011 and in CX-5279 instead of a pyridine (as in CX-4945) ring is likely to account for the higher specificity of these compounds whose Gini coefficients, calculated by profiling them against panels of 102 and/or 235 kinases, are significantly higher than that of CX-4945 (0.735 and 0.755, respectively, vs 0.615), marking the highest selectivity ever reported for CK2 inhibitors.