Synthesis of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazines that have antibacterial activity and also inhibit inorganic pyrophosphatase.

Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

Crystal structure and characterization of coiled-coil domain of the transient receptor potential channel PKD2L1.

The cation-permeable channel PKD2L1 forms a homomeric assembly as well as heteromeric associations with both PKD1 and PKD1L3, with the cytoplasmic regulatory domain (CRD) of PKD2L1 often playing a role in assembly and/or function. Our previous work indicated that the isolated PKD2L1 CRD assembles as a trimer in a manner dependent on the presence of a proposed oligomerization domain. Herein we describe the 2.7Å crystal structure of a segment containing the PKD2L1 oligomerization domain which indicates that trimerization is driven by the ß-branched residues at the first and fourth positions of a heptad repeat (commonly referred to as "a" and "d") and by a conserved R-h-x-x-h-E salt bridge motif that is largely unique to parallel trimeric coiled coils. Further analysis of the PKD2L1 CRD indicates that trimeric association is sufficiently strong that no other species are present in solution in an analytical ultracentrifugation experiment at the lowest measurable concentration of 750nM. Conversely, mutation of the "a" and "d" residues leads to formation of an exclusively monomeric species, independent of concentration. Although both monomeric and WT CRDs are stable in solution and bind calcium with 0.9µM affinity, circular dichroism studies reveal that the monomer loses 25% more α-helical content than WT when stripped of this ligand, suggesting that the CRD structure is stabilized by trimerization in the ligand-free state. This stability could play a role in the function of the full-length complex, indicating that trimerization may be important for both homo- and possibly heteromeric assemblies of PKD2L1.

Identification of the structural motif responsible for trimeric assembly of the C-terminal regulatory domains of polycystin channels PKD2L1 and PKD2.

Polycystin 2-type cation channels PKD2 and PKD2L1 interact with polycystin 1-type proteins PKD1 and PKD1L3 respectively, to form receptor-cation-channel complexes. The PKD2L1-PKD1L3 complex perceives sour taste, whereas disruption of the PKD2-PKD1 complex, responsible for mechanosensation, leads to development of ADPKD (autosomal-dominant polycystic kidney disease). Besides modulating channel activity and related signalling events, the CRDs (C-terminal regulatory domains) of PKD2 and PKD2L1 play a central role in channel oligomerization. The present study investigates the aggregation state of purified full-length PKD2L1-CRD as well as truncations of CRDs from PKD2 channels. Far- and near-UV CD spectroscopy show that the full-length PKD2L1 CRD (PKD2L1-198) and the truncated PKD2 CRD (PKD2-244) are alpha-helical with no beta-sheet, the alpha-helix content agrees with sequence-based predictions, and some of its aromatic residues are in an asymmetric environment created at least by partially structured regions. Additionally, the CRD truncations exhibit an expected biochemical function by binding Ca2+ in a physiologically relevant range with Kd values of 2.8 muM for PKD2-244 and 0.51 muM for PKD2L1-198. Complimentary biophysical and biochemical techniques establish that truncations of the PKD2 and PKD2L1 CRDs are elongated molecules that assemble as trimers, and the trimeric aggregation state is independent of Ca2+ binding. Finally, we show that a common coiled-coil motif is sufficient and necessary to drive oligomerization of the PKD2 and PKD2L1 CRD truncations under study. Despite the moderate sequence identity (39%) between CRDs of PKD2 and PKD2L1, they both form trimers, implying that trimeric organization of CRDs may be true of all polycystin channels.