Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.

Design and synthesis of prodrugs of promising drug candidates represents a valid strategy to overcome the lack of favorable ADME properties, in particular aqueous solubility and bioavailability. We report herein the successful application of this strategy with two representative pyrazolo[3,4-d]pyrimidine derivatives (1 and 2), which led to the development of the corresponding and highly water-soluble antitumor prodrugs (7 and 8). In vitro studies confirmed a significant improvement of aqueous solubility and, for compound 8, good plasma stability, suggesting superior in vivo bioavailability. As expected, the uncleaved water-soluble prodrugs 7 and 8 showed no activity toward the enzymatic targets (c-Src and c-Abl) but revealed promising antiproliferative activity in myeloid cell lines, as a consequence of the in vitro hydrolysis of the selected solubilizing moiety, followed by the release of the active compounds (1 and 2).

Cystic fibrosis (CF) is a multi-organ protein misfolding disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses, bacteria and their co-infections are recognized triggers of acute pulmonary exacerbations, accelerating disease progression, and increasing hospitalization and mortality rate. Treatment complexity increases with the age of patients, as do the number and severity of side effects, drug-drug interactions and costs. Simplifying the therapeutic regimen represents therefore one of the key priorities of CF treatment. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as mild correctors and broad-spectrum picornavirus inhibitors. Starting from the previously identified multitarget hits, we report herein the synthesis and biological profiling of new bithiazole derivatives to elucidate the structural requirements to improve F508del-CFTR correction and antiviral potencies. The most promising compound 23a inhibited PI4KIIIβ and selected picornaviruses (EV71, CVB3, hRV02), showed good F508del-CFTR correction potency, additivity and possible synergy with lumacaftor (VX809) at low micromolar concentration. In addition, it was well tolerated in vivo by C57BL/6 mice with no sign of acute toxicity and histological alterations in key biodistribution organs.

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