Buffer-free high pH mobile phase LC-MS/MS for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 20 drugs and metabolites in whole blood.

Background: Acidic mobile phases are commonly used in reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalysis. However, increased sensitivity, improved peak symmetry, and increased retention, especially for basic hydrophilic drugs have been observed using basic mobile phases. In our previous acidic mobile phase LC-MS/MS method we needed two injections (0.

Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits.

Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PP), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase).

Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway.

Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes.

Pharmacological chaperone for α-crystallin partially restores transparency in cataract models.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro.

Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries.

Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics.

Narrow-spectrum inhibitors of Campylobacter jejuni flagellar expression and growth.

Campylobacter jejuni is a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella of C. jejuni as a major determinant of chicken colonization.

The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair.

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines.

Inhibitors of TonB function identified by a high-throughput screen for inhibitors of iron acquisition in uropathogenic Escherichia coli CFT073.

The urinary tract is one of the most common sites of infection in humans, and uropathogenic Escherichia coli (UPEC) is the main causative agent of urinary tract infections. Bacteria colonizing the urinary tract face extremely low iron availability. To counteract this, UPEC expresses a wide variety of iron acquisition systems.

CYLD deubiquitinates RIP1 in the TNFα-induced necrosome to facilitate kinase activation and programmed necrosis.

Background: Necroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome.

Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis.

Programmed necrosis or necroptosis is controlled by the action of two serine/threonine kinases, RIP1 (receptor-interacting serine/threonine protein kinase 1; also known as RIPK1) and RIP3. The phosphorylation of RIP1 and RIP3 is critical for assembly of the necrosome, an amyloid-like complex that initiates transmission of the pro-necrotic signal. In the present study, we used site-directed mutagenesis to systematically examine the effects of putative phosphoacceptor sites on RIP1 and RIP3 on TNF (tumour necrosis factor)-induced programmed necrosis.

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR.

RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation.

Background: Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis.

Functional complementation between FADD and RIP1 in embryos and lymphocytes.

FADD is a common adaptor shared by several death receptors for signalling apoptosis through recruitment and activation of caspase 8 (refs 1-3). Death receptors are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, Fadd(-/-) mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ.

Bicinchoninic acid (BCA) assay in low volume.

The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates.

Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase.

Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro.

High-throughput screen for Escherichia coli heat shock protein 70 (Hsp70/DnaK): ATPase assay in low volume by exploiting energy transfer.

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects.

High-throughput screening of the virulence regulator VirF: a novel antibacterial target for shigellosis.

Shigella flexneri is a human enteropathogen that infects about 165 million people and claims more than 1 million lives per year worldwide. Although shigellosis has been considered a disease of the "Third World," like many other contagious diseases, it does occur in developed countries. The emergence of drug and multidrug-resistant strains of Shigella emphasizes the need for novel antibiotic development.

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis.

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PP(i)) exchange assays.

Inhibition of the early phase of HIV replication by an isothiazolone, PD 161374.

A new class of substituted 2'-benzisothiazolone represented by PD 161374 was discovered with antiviral activity against retroviruses similar to previously described nucleocapsid inhibitor PD 159206 (DIBA-4). In T cell culture, the 50% inhibitory concentrations (EC(50)) of PD 161374 and PD 159206 were on average 2.5 microM (ranges of 1.

2,2'-Dithiobisbenzamides and 2-benzisothiazolones, two new classes of antiretroviral agents: SAR and mechanistic considerations.

Substituted 2,2'-dithiobisbenzamides and 2-benzisothiazolones were prepared and shown to possess low microM activity with high therapeutic indices against HIV-1, HIV-2 and SIV in cell culture. The mechanism of antiviral action was determined to be directed toward the nucleocapsid protein (NCp7), which contains two zinc fingers and plays vital roles in the viral life cycle. The "active sulfides" of this study cause the extrusion of zinc from these zinc fingers.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein zinc ejection activity of disulfide benzamides and benzisothiazolones: correlation with anti-HIV and virucidal activities.

It has been shown previously by our group and others that a series of four disulfide benzamides with cellular anti-human immunodeficiency virus (HIV) activity can eject zinc from HIV type 1 nucleocapsid protein (NCp7) in vitro while analogs without antiviral activity do not. We also found that the zinc ejection activity correlates with the loss of the ability of NCp7 to bind to HIV psi RNA in vitro. These observations indicate that the antiviral disulfide benzamides may act at a novel retroviral target of action, i.

Discovery and optimization of nonpeptide HIV-1 protease inhibitors.

Several small, achiral nonpeptide inhibitors of HIV-1 protease with low micromolar activity were identified by mass screening of the Parke-Davis compound library. Two of the compounds, structurally similar, were both found to be competitive and reversible inhibitors [compound 1, 4-hydroxy-3-(3-phenoxypropyl)-1-benzopyran-2-one: Ki = 1.0 microM; compound 2, 4-hydroxy-6-phenyl-3-(phenylthio)-pyran-2-one: Ki = 1.