Eurico J. Cabrita

Non-steroidal anti-inflammatory drugs exert their pharmacological activity through inhibition of cyclooxygenase 1 and 2 (COX-1 and COX-2). Recent research suggests that a balanced inhibition of both COX-1 and COX-2 is the key to reduce the side-effects exhibited by COX inhibitors. We developed new benzimidazole-based compounds that showed a balanced COX inhibition, supported by molecular docking screening. The human whole blood assays demonstrated that the ester derivatives were potent inhibitors. Competitive saturation transfer difference (STD)-NMR experiments, in the presence of COX-2, using naproxen and diclofenac demonstrated that ester derivatives do not compete with diclofenac for the same binding site, but compete with the allosteric inhibitor naproxen. Combination of NMR spectroscopy with molecular docking has permitted us to detect a new naproxen-like inhibitor, which could be used for future drug development.

The close structural similarity between the two cyclooxygenase (COXs) isoforms and the absence of selective inhibitors without side effects continues to stimulate the development of novel approaches towards selective anti-inflammatory drugs. In the present study a small library of new indolic compounds involving two different substitutions patterns at the indole scaffold was synthesized. In order to establish a relation between the spatial distribution of known functional groups related with inhibitory activity, two substitution patterns were explored: one with substituents at N-1, C-3, C-5 positions and another at C-2, C-3 and C5 positions. Accordingly, indole positions C-5, C-3 and N-1 were substituted with: sulfonamide or methylsulfone at C-5, p-halo-benzyl group at C-3, and an alkyl chain with a trifluoromethyl group at N-1. Alternatively, a p-halo-benzyl group was introduced at C-2, leaving the indolic nitrogen free. Inhibitory studies were performed and the activity results obtained against both COXs isoforms were rationalized based on docking and NMR studies. Docking studies show that dialkyation at C-2 and C-3 favors a binding with an orientation similar to that of the known selective inhibitor SC-558. From the tested compounds, this substitution pattern is correlated with the highest inhibitory activity and selectivity: 70% COX-2 inhibition at 50 M, and low COX-1 inhibition (18±9%). Additionally, Saturation Transfer Difference NMR experiments reveal different interaction patterns with both COXs isoforms that may be related with different orientations of the sulfonamide group in the binding pocket. Despite the moderated inhibitory activities found, this study represents an innovative approach towards COXs inhibitory activity rationalization and to the design of anti-inflammatory drugs.

This paper describes for the first time the intimate molecular details of the association between a platinated oligonucleotide and a zinc-finger peptide. Site-specific platination of the guanine in a ss hexanucleotide gave {[Pt(dien)d(5’-TACGCC-3’)], Pt(dien)(6-mer)}, II, characterized by mass spectrometry and 1H-NMR spectroscopy. The work extends the study of platinum-nucleobase complex-zinc finger interactions using small molecules such as [Pt(dien)(9-EtGua)]2+, I . The structure of the (34-52) C-terminal finger of the HIV nucleocapsid protein HIVNCp7 (ZF1) was characterized by 1H-NMR spectroscopy and compared with that of the N-terminal single finger and the 2-finger “intact” NCp7. Interaction of II with ZF1 results in significant changes in comparison to the “free” uncomplexed hexanucleotide – the major shifts occur for Trp37 resonances are broadened and shifted upfield and other major shifts are for Gln45 (H21, H3, Q), Met46 (NH, H2), Lys47 (NH, Q) and Glu50 (H2, H3). The Zn-Cys/His chemical shifts show only marginal deviations. The solution structure of ZF1, the 6-mer/ZF1 and II/ZF1 adducts were calculated from the NOESY-derived distance constraints. The DNA position in II/ZF1 is completely different than in the absence of platinum. Major differences are the appearance of new Met46-Cyt6H5 and Trp37-Cyt5H5 contacts but severe weakening of the Trp37-Gua4 contact, attributed to the steric effects caused by Gua4 platination, accompanied by a change in the position of the aromatic ring. The results demonstrate the feasibility of targetting specific ZF motifs with DNA-tethered coordination compounds, such as Pt compounds and Co-macrocycles – with implications for drug targetting and indeed the intimate mechansims of DNA repair of platinated DNA.