Kinase inhibition continues to be a major focus of pharmaceutical research and discovery due to the central role of these proteins in the regulation of cellular processes. One family of kinases of pharmacological interest, due to its role in activation of immunostimulatory pathways, is the Janus kinase family. Small molecule inhibitors targeting the individual kinase proteins within this family have long been sought-after therapies.
View Article and Find Full Text PDFA riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC = 0.3 μM), supported that a component of the riboflavin synthesis pathway was the molecular target.
View Article and Find Full Text PDFRiboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli.
View Article and Find Full Text PDFCoagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions.
View Article and Find Full Text PDFBacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway.
View Article and Find Full Text PDFThis review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds.
View Article and Find Full Text PDFRiboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors.
View Article and Find Full Text PDFOligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea.
View Article and Find Full Text PDFThe OmpR/PhoB family of response regulators (RRs) is the largest class of two-component system signal transduction proteins. Extensive biochemical and structural characterization of these transcription factors has provided insights into their activation and DNA-binding mechanisms. For the most part, OmpR/PhoB family proteins are thought to become activated through phosphorylation from their cognate histidine kinase partners, which in turn facilitates an allosteric change in the RR, enabling homodimerization and subsequently enhanced DNA binding.
View Article and Find Full Text PDFSingle-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain.
View Article and Find Full Text PDFDNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains.
View Article and Find Full Text PDFThe LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.
View Article and Find Full Text PDFOxazole-containing macrocycles, which include the natural product telomestatin, represent a promising class of anticancer agents that target G-quadruplex DNA. Two synthetic hexaoxazole-containing macrocyclic compounds (HXDV and HXLV-AC) have been characterized with regard to their cytotoxic activities versus human cancer cells, as well as the mode, thermodynamics, and specificity with which they bind to the intramolecular (3+1) G-quadruplex structural motif formed in the presence of K+ ions by human telomeric DNA. Both compounds exhibit cytotoxic activities versus human lymphoblast (RPMI 8402) and oral carcinoma (KB3-1) cells, with associated IC50 values ranging from 0.
View Article and Find Full Text PDFSpectroscopic and calorimetric techniques have been employed to characterize the impact of incorporation of the fluorescent base analog 2-aminopurine into the 1492 or 1493 position of an E. coli rRNA A-site model oligonucleotide, as well as the energetics and dynamics associated with recognition of this A-site model oligomer by aminoglycoside antibiotics. The results of these studies indicate that incorporation of 2AP into either the 1492 or 1493 position does not perturb the structure or stability of the host RNA or its aminoglycoside binding affinity.
View Article and Find Full Text PDFRecent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis.
View Article and Find Full Text PDFThe lack of absolute prokaryotic selectivity of natural antibiotics is widespread and is a significant clinical problem. The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases is extremely challenging. Here, we have used a combination of biochemical and structural analysis to compare and contrast the molecular mechanisms of action and the structure-activity relationships of a new synthetic aminoglycoside, NB33, and a structurally similar natural aminoglycoside apramycin.
View Article and Find Full Text PDFOxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form.
View Article and Find Full Text PDFThe growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance.
View Article and Find Full Text PDF2-Deoxystreptamine (2-DOS) aminoglycosides exert their antibiotic actions by binding to the A site of the 16S rRNA and interfering with bacterial protein synthesis. However, the molecular forces that govern the antitranslational activities of aminoglycosides are poorly understood. Here, we describe studies aimed at elucidating these molecular forces.
View Article and Find Full Text PDFSeveral G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture.
View Article and Find Full Text PDFThe terbenzimidazoles are a class of anticancer agents that bind in the DNA minor groove. These compounds also exhibit a propensity for self-association, which can potentially impact their cellular bioavailabilities and activities. We have explored this possibility by using a broad range of biophysical and cytological techniques to characterize the self-association and cellular uptake properties of two terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB) and 5-phenyl-2'-(indolo-6-yl)bibenzimidazole (5P2'IBB).
View Article and Find Full Text PDFSteady-state and time-resolved fluorescence techniques have been used to characterize the energetics and dynamics associated with the interaction of an E. coli 16 S rRNA A-site model oligonucleotide and four aminoglycoside antibiotics that exhibit a broad range of antibacterial activity. The results of these characterizations suggest that aminoglycoside-induced reduction in the mobility of an adenine residue at position 1492 of the rRNA A-site is a more important determinant of antibacterial activity than drug affinity for the A-site.
View Article and Find Full Text PDFThe binding of aminoglycoside antibiotics to a broad range of macromolecular targets is coupled to protonation of one or more of the amino groups that typify this class of drugs. Determining how and to what extent this linkage influences the energetics of the aminoglycoside-macromolecule binding reaction requires a detailed understanding of the thermodynamics associated with the protonation equilibria of the aminoglycoside amino groups. In recognition of this need, a calorimetric- and NMR-based approach for obtaining the requisite thermodynamic information is presented using paromomycin as the model aminoglycoside.
View Article and Find Full Text PDF2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics exert their antimicrobial activities by targeting the decoding region A site of the rRNA and inhibiting protein synthesis. A prokaryotic specificity of action is critical to therapeutic utility of 2-DOS aminoglycosides as antibiotics. Here, isothermal titration calorimetry (ITC) and fluorescence studies are presented that provide insight into the molecular basis for this prokaryotic specificity of action.
View Article and Find Full Text PDFIsothermal titration calorimetry (ITC), computational, and osmotic stress techniques have been used to characterize the changes in heat capacity, solvent-accessible surface, and hydration that accompany the binding of the aminoglycoside paromomycin to both prokaryotic and eukaryotic rRNA A-site model oligonucleotides. Regarded as a whole, the results of these studies suggest that the intrinsic heat capacity change (DeltaC(p)) for the binding of paromomycin to each rRNA A-site is near zero, with the negative DeltaC(p) observed for the binding of the drug to the prokaryotic rRNA A-site being dictated by the coupled destacking of the adenine residues at positions 1492 and 1493. In this connection, DeltaC(p) provides a useful calorimetric signature for assessing the relative impacts of novel and existing A-site targeting ligands on rRNA conformation, which, in turn, should provide a useful analytical tool for facilitating the drug design process, since aminoglycoside-induced destacking of A1492 and A1493 is thought to be a determining factor in the mistranslational and antimicrobial activities of the drugs.
View Article and Find Full Text PDF