Selective killing of nonreplicating mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.     

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel alpha1 subunit

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an alpha(2)delta subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced "on-off" transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic alpha(1) subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the alpha(2)delta Ca(2+) channel subunit in mediating the proper localization of an alpha(1) subunit at synapses.     

Evolutionary Relationships of Primary Prokaryotic Endosymbionts of Whiteflies and Their Hosts

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are plant sap-sucking insects that harbor prokaryotic primary endosymbionts (P-endosymbionts) within specialized cells located in their body cavity. Four-kilobase DNA fragments containing 16S-23S ribosomal DNA (rDNA) were amplified from the P-endosymbiont of 24 whiteflies from 22 different species of 2 whitefly subfamilies. In addition, 3-kb DNA fragments containing mitochondrial cytB, nd1, and large-subunit rDNA (LrDNA) were amplified from 17 whitefly species. Comparisons of the P-endosymbiont (16S-23S rDNA) and host (cytB-nd1-LrDNA) phylogenetic trees indicated overall congruence consistent with a single infection of a whitefly ancestor with a bacterium and subsequent cospeciation (cocladogenesis) of the host and the P-endosymbiont. On the basis of both the P-endosymbiont and host trees, the whiteflies could be subdivided into at least five clusters. The major subdivision was between the subfamilies Aleyrodinae and Aleurodicinae. Unlike the P-endosymbionts of may other insects, the P-endosymbionts of whiteflies were related to Pseudomonas and possibly to the P-endosymbionts of psyllids. The lineage consisting of the P-endosymbionts of whiteflies is given the designation “Candidatus Portiera” gen. nov., with a single species, “Candidatus Portiera aleyrodidarum” sp. nov.     

Novel imidazole-based histamine H3 antagonists

A novel series of imidazole containing histamine H₃ receptor ligands were investigated and found to be potent functional antagonists. After improving the stability of these molecules towards liver microsomes, these compounds were found to have no appreciable affinity for CYP P450s. Subsequent in vivo experiments showed significant brain uptake of (4-chloro-phenyl)-[2-(1-isopropyl-piperidin-4-ylmethoxy)-3-methyl-3H-imidazol-4-yl]-methanone 22.     

Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.     

Imidazo[1,2‐c]pyrimidin‐5(6H)‐one as a novel core of cyclin‐dependent kinase 2 inhibitors: Synthesis,activity measurement,docking, and quantum mechanical scoring

We report on the synthesis, activity testing, docking, and quantum mechanical scoring of novel imidazo[1,2‐c]pyrimidin‐5(6H)‐one scaffold for cyclin‐dependent kinase 2 (CDK2) inhibition. A series of 26 compounds substituted with aromatic moieties at position 8 has been tested in in vitro enzyme assays and shown to inhibit CDK2. 2D structure‐activity relationships have ascertained that small substituents at position 8 (up to the size of naphtyl or methoxyphenyl) generally lead to single‐digit micromolar IC₅₀ values, whereas bigger substituents (substituted biphenyls) decreased the compounds' activities. The binding modes of the compounds obtained using Glide docking have exhibited up to 2 hinge‐region hydrogen bonds to CDK2 and differed in the orientation of the inhibitor core and the placement of the 8‐substituents. Semiempirical quantum mechanics‐based scoring identified probable favourable binding modes, which will serve for future structure‐based design and synthetic optimization of substituents of the heterocyclic core. In summary, we have identified a novel core for CDK2 inhibition and will explore it further to increase the potencies of the compounds and also monitor selectivities against other protein kinases.