Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX

The enzyme organophosphorus acid anhydrolase (OPAA), from <i>Alteromonas</i> sp. JD6.5, has been shown to rapidly catalyze the hydrolysis of a number of toxic organophosphorus compounds, including several G-type chemical nerve agents. The enzyme was cloned into <i>Escherichia coli</i> and can be produced up to approximately 50% of cellular protein. There have been no previous reports of OPAA activity on VR {Russian VX, <i>O</i>-isobutyl <i>S</i>-[2-(diethylamino)­ethyl] methylphosphonothioate}, and our studies reported here show that wild-type OPAA has poor catalytic efficacy toward VR. However, via application of a structurally aided protein engineering approach, significant improvements in catalytic efficiency were realized via optimization of the small pocket within the OPAA’s substrate-binding site. This optimization involved alterations at only three amino acid sites resulting in a 30-fold increase in catalytic efficiency toward racemic VR, with a strong stereospecificity toward the P­(+) enantiomer. X-ray structures of this mutant as well as one of its predecessors provide potential structural rationales for their effect on the OPAA active site. Additionally, a fourth mutation at a site near the small pocket was found to relax the stereospecificity of the OPAA enzyme. Thus, it allows the altered enzyme to effectively process both VR enantiomers and should be a useful genetic background in which to seek further improvements in OPAA VR activity

Insights into the Porcine Reproductive and Respiratory Syndrome Virus Viral Ovarian Tumor Domain Protease Specificity for Ubiquitin and Interferon Stimulated Gene Product 15

Porcine reproductive and respiratory syndrome (PRRS) is a widespread economically devastating disease caused by PRRS virus (PRRSV). First recognized in the late 1980s, PRRSV is known to undergo somatic mutations and high frequency viral recombination, which leads to many diverse viral strains. This includes differences within viral virulence factors, such as the viral ovarian tumor domain (vOTU) protease, also referred to as the papain-like protease 2. These proteases down-regulate innate immunity by deubiquitinating proteins targeted by the cell for further processing and potentially also acting against interferon-stimulated genes (ISGs). Recently, vOTUs from vaccine derivative Ingelvac PRRS modified live virus (MLV) and the highly pathogenic PRRSV strain JXwn06 were biochemically characterized, revealing a marked difference in activity toward K63 linked polyubiquitin chains and a limited preference for interferon-stimulated gene product 15 (ISG15) substrates. To extend our research, the vOTUs from NADC31 (low virulence) and SDSU73 (moderately virulent) were biochemically characterized using a myriad of ubiquitin and ISG15 related assays. The K63 polyubiquitin cleavage activity profiles of these vOTUs were found to track with the established pathogenesis of MLV, NADC31, SDSU73, and JXwn06 strains. Fascinatingly, NADC31 demonstrated significantly enhanced activity toward ISG15 substrates compared to its counterparts. Utilizing this information and strain–strain differences within the vOTU encoding region, sites were identified that can modulate K63 polyubiquitin and ISG15 cleavage activities. This information represents the basis for new tools to probe the role of vOTUs in the context of PRRSV pathogenesis

A Noncompetitive Inhibitor for <i>Mycobacterium tuberculosis</i>’s Class IIa Fructose 1,6-Bisphosphate Aldolase

Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as <i>Mycobacterium tuberculosis</i>, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC₅₀ of 10 ± 1 μM for the class II FBA present in <i>M. tuberculosis</i> (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant <i>Staphylococcus aureus</i>. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates

Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from <i>Mycobacterium tuberculosis</i>

Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn­(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from <i>Mycobacterium tuberculosis</i> (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form

Development and validation of a yeast high-throughput screen for inhibitors of A{beta}42 oligomerization

Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid β (Aβ), as the primary toxic species in Alzheimer’s disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aβ42 oligomer formation. Specifically, Aβ42 fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aβ42 oligomerization

Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles

<div><p>Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the <i>Bunyaviridae</i> family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.</p></div

CCHFV strain IbAr10200 replication machinery does not significantly discriminate against minigenomes from other CCHFV strains.

<p>(A) Absolute NanoLuc signal in relative light units (RLU) in BSR-T7 cells transfected with IbAr10200 NP, L, and GPC plasmids, and with minigenomes encoding L NCR of other CCHFV strains, as indicated. Data are reported as standard error of the mean (n = 8 from 2 experiments). (B) TCID₅₀ determination in SW-13 cells treated with supernatants from cells in (A). Results from 2 experiments are shown. (C) NanoLuc signal in SW-13 cells treated as in (B) (n = 8 from 2 experiments). NanoLuc signal data in VLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP, L and the corresponding minigenome. All data are reported as standard error of the mean.</p

Exchanging surface glycoproteins has a greater impact on tecVLP titer and NanoLuc signal than exchanging minigenomes.

<p>(A) TCID₅₀ titers/mL and NanoLuc signals in relative light units (RLU) measured in SW-13 cells treated with tecVLPs with Afg09 GPC and with L segment NCR minigenomes from indicated CCHFV strains. NanoLuc data are presented as standard error of the mean (average of 3 experiments), and results of 3 TCID₅₀ experiments are shown. (B) TCID₅₀ titers/mL and NanoLuc signals measured in SW-13 cells treated with tecVLPs with Oman GPC and L segment NCR minigenomes from indicated CCHFV strains. NanoLuc signal data in tecVLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP, L and the corresponding minigenome. NanoLuc data are reported as standard error of the mean (average of 3 experiments), and results of 3 TCID₅₀ experiments are shown.</p

CCHFV tecVLP morphology and glycoprotein-mediated entry.

<p>(A) Representative electron microscopy images of tecVLP. (B) Neutralization of tecVLP entry into SW-13 cells by previously reported monoclonal antibodies (mAbs). 11E7 and 12A9 are CCHFV neutralizing antibodies targeting the glycoprotein Gc; 13G8 is a non-neutralizing mAb targeting the immature glycoprotein PreGn; 9D5 targets the NP protein. Data are displayed as standard error of the mean (average of 2 experiments).</p

Organization and signal expression of the Crimean-Congo hemorrhagic fever virus (CCHFV) minigenome and support plasmids in transfected cells, and in cells treated with transcription- and entry-competent virus-like particles (tecVLP).

<p>(A) Basic organization of CCHFV helper plasmids and minigenomes used in these experiments. Helper plasmids encoding the nucleocapsid protein (NP), the viral RNA-dependent RNA-polymerase (L), and glycoprotein precursor (GPC) are under the control of the chicken β-actin polymerase II (pol II) promoter. Replication machinery genes (NP and L) are from the CCHFV strain IbAr10200, and the surface GPC are from IbAr10200, Oman, Turkey, or Afg09 CCHFV strains, as indicated. The minigenome plasmids encode the NanoLuc (Luc) reporter gene in reverse, or non-coding, sense; NanoLuc is flanked by non-coding regions (NCR) from one of the 3 CCHFV genomic segments, S, M, or L. Minigenomes of all 3 segment NCRs from strain IbAr10200, and only the L segment NCR from strains Afg09 and Oman, were used in these experiments. (B) Absolute NanoLuc signal levels, in relative light units (RLU), following transfection of BSR-T7 cells with minigenome and replication plasmids (NP + L) or minigenome and tecVLP assembly plasmids (L + NP + GPC). Data are reported as standard error of the mean. ** p < 0.01, ***p < 0.001 (average of 2 experiments). (C) Titration experiments from 2 tecVLP production experiments passaged into SW-13 cells. S, M, and L NCR minigenomes are shown. (D) NanoLuc signal (in RLU) in SW-13 cells treated with supernatants from BSR-T7 cells transfected with tecVLP assembly plasmids (average of 2 experiments). NanoLuc signal data in tecVLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP and L. All data are reported as standard error of the mean.</p