Synthesis, structural analysis, and biological evaluation of thioxoquinazoline derivatives as phosphodiesterase 7 inhibitors.

PDE7 inhibitors regulate pro-inflammatory and immune T-cell functions, and are a potentially novel class of drugs especially useful in the treatment of a wide variety of immune and inflammatory disorders. Starting from our lead family of thioxoquinazolines, we designed, synthesized, and characterized a novel series of thioxoquinazoline derivatives. Many of these compounds showed inhibitory potencies at sub-micromolar levels against the catalytic domain of PDE7A1 and at the micromolar level against PDE4D2. Cell-based studies showed that these compounds not only increased intracellular cAMP levels, but also had interesting anti-inflammatory properties within a therapeutic window. The in silico data predict that these compounds are capable of the crossing the blood-brain barrier. The X-ray crystal structure of the PDE7A1 catalytic domain in complex with compound 15 at a resolution of 2.4 A demonstrated that hydrophobic interactions at the active site pocket are a key feature. This structure, together with molecular modeling, provides insight into the selectivity of the PDE inhibitors and a template for the discovery of new PDE7 or PDE7/PDE4 dual inhibitors.

Role of Pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis.

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.

Unconventional interactions between water and heterocyclic nitrogens in protein structures.

We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.

Ligand-induced changes in the binding sites of proteins.

Classical molecular interaction potentials, in conjunction with other theoretical techniques, are used to analyze the dependence of the binding sites of representative proteins on the bound ligand. It is found that the ligand bound introduces in general small structural perturbations at the binding site of the protein. However, such small structural changes can lead to important alterations in the recognition pattern of the protein. The impact of these findings in docking procedures is discussed.

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines.

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.