Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS.

The N-terminal regions (tails) of histone proteins are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly because of their inherently disordered nature. Although hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably because of the poor N-terminal coverage provided by pepsin. Inspired from histone-clipping events, we profiled the activity of cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3, and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase.

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-ß-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Structural and conformational determinants of macrocycle cell permeability.

Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.

Conservation of the structure and function of bacterial tryptophan synthases.

Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes from Salmonella typhimurium and Escherichia coli serve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5'-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and ß (TrpB), functioning as a linear αßßα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the ß-replacement of the hydroxyl group of l-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria in macrophages and for evading host defense. Therefore, TrpAB is a promising target for drug discovery, as its orthologs include enzymes from the important human pathogens Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis, the causative agents of pneumonia, legionnaires' disease and tularemia, respectively. However, specific biochemical and structural properties of the TrpABs from these organisms have not been investigated. To fill the important phylogenetic gaps in the understanding of TrpABs and to uncover unique features of TrpAB orthologs to spearhead future drug-discovery efforts, the TrpABs from L. pneumophila, F. tularensis and S. pneumoniae have been characterized. In addition to kinetic properties and inhibitor-sensitivity data, structural information gathered using X-ray crystallo-graphy is presented. The enzymes show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors.

Novel tricyclic glycal-based TRIB1 inducers that reprogram LDL metabolism in hepatic cells.

Increased expression of the Tribbles pseudokinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction. We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518, the initial lead compound from the tricyclic glycal class, exhibited stereochemically dependent activity and the potency far exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518 demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes. Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518 stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in vivo studies.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1.

Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

Expanded electrical model of a contactless conductivity detector: development and verification.

A theoretical model of the contactless conductivity detector (CCD) has been developed consisting of a network of resistors and capacitors. The output of the model is compared to experimental results and to the output of a simpler model. Experimentally, a lock-in amplifier is added to the detection scheme of the contactless conductivity detector to provide a more sensitive method of signal isolation. The detector is assembled on a printed circuit board with the electrodes in a co-axial configuration. The electrodes are chosen to allow for use with fused silica capillaries in capillary electrophoresis. The use of a lock-in amplifier in place of a previous rectification/filtering circuit allows for an approximate 10-fold improvement in S/N. The detector shows a linear response to changes in excitation voltage and to changes in analyte concentration. Mass limits of detection of 60, 63, and 50 fg are determined for the inorganic cations potassium, sodium, and lithium, respectively (for a signal three times the level of the rms noise).

Development and validation of a stability-indicating RP-HPLC method for simultaneous assay of betamethasone dipropionate, chlorocresol, and for the estimation of betamethasone dipropionate related compounds in a pharmaceutical cream and ointment.

A new stability-indicating reversed-phase HPLC (RP-HPLC) method has been developed and validated for simultaneous assay of betamethasone dipropionate (BD) and chlorocresol and also for the estimation of BD related compounds in a pharmaceutical cream matrix. In addition, this newly developed RP-HPLC method was also demonstrated as suitable for a pharmaceutical ointment product that does not contain chlorocresol. The RP-HPLC method uses a Waters SymmetryShield RP18 analytical column (150 × 4.6 mm). Water (mobile phase A) and acetonitrile (mobile phase B) were used in the gradient elution with a flow rate of 1.5 mL/min and detection wavelength at 240 nm. A Waters XBridge Shield RP18 analytical column (150 × 4.6 mm) was identified as an alternate column. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.02 µg/mL and 0.05 µg/mL, respectively. The precision of the method for BD is less than 0.3% RSD, and the accuracy of BD ranged from 99.5% to 102.6%. The stability-indicating capability of this method has been demonstrated by analyzing aged samples of the product. This RP-HPLC method was successfully validated per ICH guidelines and proved to be suitable for routine quality control use.

Development of a conductivity-based photothermal absorbance detector for capillary separations.

A contactless conductivity-based absorbance detector has been developed for use with capillary separations. Detection is based on a photothermal process. As analytes pass through the detector they absorb light, producing a thermal perturbation. This thermal event results in a change in the solution conductivity. The measured change in conductivity is directly related to the absorption of light. The major advantage to this type of detector is that the measured absorbance is, to a first approximation, independent of optical path length, allowing small-diameter capillaries to be used. This approach combines the optical simplicity of traditional transmission-based instruments with the path length independence of similar refraction-based photothermal detectors. In addition to the initial development and characterization of the photothermal absorbance detector, multiphysical modeling of the heat transfer within the conductivity cell was performed.