Journal: Journal of Clinical Microbiology

Article Title: Optimization of a Combined Human Parechovirus-Enterovirus Real-Time Reverse Transcription-PCR Assay and Evaluation of a New Parechovirus 3-Specific Assay for Cerebrospinal Fluid Specimen Testing

doi: 10.1128/JCM.01982-12

Figure Lengend Snippet: Comparison of CT values of EV, pan-HPeV, and HPeV3 assays with CSF clinical specimens tested in one-step and two-step RT-PCR reactions. (A) Two-step EV assay with the AGP kit enzyme versus one-step Cepheid enterovirus ASR (year 2008; n = 25); (B) two-step EV assay with the AGP kit enzyme versus one-step Argene enterovirus RUO assay (year 2009; n = 25); (C) two-step versus one-step EV assay with the AGP kit enzyme (year 2012; n = 30); (D) two-step versus one-step pan-HPeV assay with the AGP kit enzyme and historic two-step pan-HPeV assay with the ABI enzyme; (E) two-step versus one-step HPeV3 assay with the AGP kit enzyme and historic two-step HPeV3 assay with the ABI enzyme.

Article Snippet: The analytical specificity of the pan-HPeV assay reagents was tested against 13 enterovirus strains and other viruses obtained from ATCC: coxsackievirus types A9 (VR-186), B1 (VR-1032), B2 (VR-29), B3 (VR-30), B4 (VR-184), and B5 (VR-185); echovirus types 3 (VR-1040), 4 (VR-1041), 5 (VR-1043), 6 (VR-36), 7 (VR-37), 9 (VR-39), and 11 (VR-41); rhinovirus types 39 (ATCC VR-340), 8 (ATCC VR-488), 13 (ATCC VR-1123), 26 (ATCC VR-1136), and 27 (ATCC VR-1137); herpes simplex virus 1 (HSV-1) (ATCC VR-539) and HSV-2 (ATCC VR-540); human herpesvirus 6 (HHV-6; ATCC VR-1480); and laboratory-grown clinical isolates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV).

Techniques: Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Activity of DHFR inhibitors against M. abscessus ATCC 19977 and its recombinant DHFR enzyme

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: Activity Assay, Recombinant

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Activity of selected DHFR inhibitors against recombinant M. abscessus ATCC 19977 DHFR enzyme. Enzyme inhibition dose-response curves are shown for ( A ) PQD-1, ( B ) trimetrexate (TMX), ( C ) WR99210, and ( D ) trimethoprim (TMP). The data were fitted to a non-linear regression curve using the variable slope model, and the IC 50 values were calculated using GraphPad Prism 9. Percent activity values are the means of three independently performed experiments, and error bars indicate standard deviations.

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: Activity Assay, Recombinant, Enzyme Inhibition Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: In silico model of PQD-1 and trimethoprim binding to M. abscessus ATCC 19977 DHFR (PDB code: 7K6C). ( A, C ) Optimized poses of PQD-1 (yellow sticks) and trimethoprim (green sticks) with interactions with DHFR active site residues. DHFR is colored in cyan, with the residues in the binding pockets shown as cyan sticks. ( B, D ) Two-dimensional presentation of the key interactions of PQD-1 ( B ) and trimethoprim ( D ) with their binding pockets shown in ( A, C ). The hydrogen bonds, salt bridges, and hydrophobic and π-π stacking interactions are depicted as green, orange, pink, and warm pink dashed lines, respectively.

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: In Silico, Binding Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Mechanism of action of PQD-1. ( A ) Effect of DHFR over-expression on PQD-1 susceptibility of M. abscessus ATCC 19977. Cultures were treated for 3 days with PQD-1 (top) or TMX (bottom), the second most potent M. abscessus inhibitor identified in the initial whole cell screen and confirmed as a biochemical inhibitor of M. abscessus DHFR . Percent growth values are the means of three independently carried out experiments, and error bars indicate standard deviations. ( B ) Schematic of mycobacterial folate pathway and role of thymidylate synthase (TYMS) ThyA. DHPS, inhibited by SMX, converts p -aminobenzoic acid (pABA) into dihydropteroate (DHP), which in turn is converted to dihydrofolate (DHF). DHFR, inhibited by PQD-1, reduces DHF to tetrahydrofolate (THF), which is converted into methyl-tetrahydrofolate (mTHF). The TYMS ThyA catalyzes the reductive methylation of deoxyuridine-monophosphate (dUMP) to deoxythymidine-monophosphate (dTMP) utilizing mTHF as the methyl donor and reductant in the reaction, yielding DHF, the substrate of DHFR, as a by-product. ThyX is a second thymidylate synthase catalyzing the reductive methylation of dUMP to dTMP utilizing mTHF only as the methyl donor (hence generating THF instead of DHF) and NADPH as the reductant. The pathway is according to Hajian et al. . ( C ) Plot of the fractional inhibitory concentrations (FIC) of PQD-1 (top) and TMX (bottom) versus DHPS inhibitor SMX. The FIC index (FICI), calculated as (MIC A combi /MIC A alone ) + (MIC B combi /MIC B alone ), indicates synergy if <0.5. The FICs were calculated based on the MIC 50 values. The experiments were carried out three times independently in duplicate, and the presented data are one representative example.

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: Over Expression, Methylation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Characterization of spontaneous PQD-1 resistant M. abscessus ATCC 19977 strains ^a

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Dynamic SAR of PQD-1 analogs against M. abscessus ATCC 19977 and its recombinant DHFR enzyme

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: Recombinant

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus

doi: 10.1128/aac.00717-23

Figure Lengend Snippet: Activity of PQD-1 against M. abscessus subspecies reference strains and clinical isolates

Article Snippet: To identify a DHFR inhibitor with whole cell activity against M. abscessus , three DHFR inhibitors previously shown to be active against M. tuberculosis were tested against the reference strain M. abscessus subsp. abscessus ATCC 19977: the pyrrolo-quinazoline PQD-1 , the methyl-quinazoline trimetrexate , and the triazine WR99210 ( , ) ( ).

Techniques: Activity Assay