LC-MS/MS Validation for Drug of Abuse Testing Utilizing a Split Sample Oral Fluid Collection System.

The Substance Abuse and Mental Health Services Administration (SAMHSA) recently authorized oral fluid (OF) as a preferable biofluid for drugs of abuse (DOA) screening compared to urine, and they required that each screening method be confirmed by a laboratory test. We developed a DOA mass spectrometry (MS) assay optimized for undiluted OF as a matching confirmatory test for the EZ-Saliva point of care (POC), split sample, rapid visual test. Using a double isotope ratio standardization, we achieved a limit of detection of 2 = 0.99, lack of interference (90%). Stability from degradation exceeded 72 hours. The lateral flow immunoassay strips of the POC exhibited a dose-dependent response, with a 90% sensitivity and 100% specificity for N=22 self-reported, THC patient OF, digitized for quantitation. We conclude that the split sample POC device in combination with the MS assay meets the SAMHSA stated requirements for a POC test with a laboratory confirmation. Split sample collection has significant advantages because it minimizes potential error created by taking a separate OF sample for laboratory confirmation. We recommend scaling to a larger validation study set and quantification of user OF THC levels that correlate with driver impairment levels. </p

Drug discovery efforts at George Mason University

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena.To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents.To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration.To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B.Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics.The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the &ldquo;divide and conquer&rdquo; algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 &Aring; threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 &Aring; constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions

A secretory form of Parkin‐independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo

Abstract We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP‐4T1 syngeneic murine cancer model to study EVs in‐transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV‐associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission‐induced mitophagy preferentially populated the CD81+/PD‐L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin‐independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC‐1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti‐CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology

Synthesis and Bioactivity of beta-Substituted Fosmidomycin Analogues Targeting 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the beta-position of the hydroxamate analogue of 2. While direct addition of a beta-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the longer compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamates methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth

Kinetic Characterization and Allosteric Inhibition of the <i>Yersinia pestis</i> 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

<div><p>The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from <i>Yersinia pestis</i>. Enzyme assays indicate apparent kinetic constants of K_M^DXP = 252 µM and K_M^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the <i>Y. pestis</i> MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.</p></div