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1.
J Pediatric Infect Dis Soc ; 10(2): 104-111, 2021 Mar 26.
Article in English | MEDLINE | ID: mdl-32170944

ABSTRACT

BACKGROUND: Individual pharmacokinetic variability is a driver of poor tuberculosis (TB) treatment outcomes. We developed a method for measurement of rifampin concentrations by urine colorimetry and a mobile phone photographic application to predict clinically important serum rifampin pharmacokinetic measurements in children treated for TB. METHODS: Among spiked urine samples, colorimetric assay performance was tested with conventional spectrophotometric and the mobile phone/light box methods under various environmental and biologic conditions. Urine rifampin absorbance (Abs) was then determined from timed specimens from children treated for TB in Tanzania, and compared to serum pharmacokinetic measurements collected throughout the dosing interval. RESULTS: Both the mobile phone/light box and spectrophotometry demonstrated excellent correlation across a wide range of urine rifampin concentrations (7.8-1000 mg/L) in intra- and interday trials, 24-hour exposure to ambient light or darkness, and varying urinalysis profiles (all r ≥ 0.98). In 12 Tanzanian children, the urine mobile phone/light box measurement and serum peak concentration (Cmax) were significantly correlated (P = .004). Using a Cmax target of 8 mg/L, the area under the receiver operating characteristic curve was 80.1% (range, 47.2%-100%). A urine mobile phone/light box threshold of 50 Abs correctly classified all patients (n = 6) with serum measurements below target. CONCLUSIONS: The urine colorimetry with mobile phone/light box assay accurately measured rifampin absorbance in varying environmental and biological conditions that may be observed clinically. Among children treated for TB, the assay was sensitive for detection of low rifampin serum concentrations. Future work will identify the optimal timing for urine collection, and operationalize use in TB-endemic settings.


Subject(s)
Cell Phone , Tuberculosis , Antitubercular Agents/therapeutic use , Child , Colorimetry , Humans , Rifampin/therapeutic use , Tuberculosis/diagnosis , Tuberculosis/drug therapy
2.
Front Pharmacol ; 11: 1103, 2020.
Article in English | MEDLINE | ID: mdl-32848735

ABSTRACT

BACKGROUND: The potential for hepatotoxicity during isoniazid-based tuberculosis (TB) treatment presents a major challenge for TB control programs worldwide. We sought to determine whether pharmacokinetic exposures of isoniazid and its metabolites were related to cellular oxidation/reduction status and downstream markers of oxidative DNA damage. METHODS: We performed intensive pharmacokinetic sampling among isoniazid-treated patients to determine the relative plasma exposures of isoniazid, acetylisoniazid, hydrazine, and acetylhydrazine. Physiologically-based pharmacokinetic modeling was used to estimate liver tissue exposures during a 24-h dosing interval for each compound. We experimentally treated HepG2 cells with isoniazid and metabolites at equimolar concentrations corresponding to these exposures for 7, 14, and 28-day periods, and performed assays related to redox imbalance and oxidative DNA damage at each timepoint. We related a urine marker of oxidative DNA damage to serum isoniazid pharmacokinetic exposures and pharmacogenetics in a clinical study. RESULTS: Among isoniazid-treated patients, serum concentrations of hydrazine and isoniazid concentrations were highly correlated. At equimolar concentrations that approximated hepatic tissue exposures during a 24-h dosing interval, hydrazine demonstrated the highest levels of redox imbalance, mitochondrial injury, and oxidative DNA damage over a 28-day treatment period. In a clinical validation study of isoniazid-treated TB patients, peak isoniazid serum concentrations were positively associated with a urine biomarker of oxidative DNA damage. CONCLUSIONS: Isoniazid and its metabolites share the potential for oxidative cellular damage, with the greatest effects observed for hydrazine. Future studies should investigate the clinical consequences of oxidative stress with regards to clinical episodes of drug induced liver injury during isoniazid treatment.

3.
Int J Infect Dis ; 84: 15-21, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31051278

ABSTRACT

OBJECTIVE: There is considerable uncertainty regarding the optimal use of rifampicin for the treatment of tuberculous (TB) meningitis. A pharmacokinetic modeling and simulation study of rifampicin concentrations in cerebrospinal fluid (CSF) during TB meningitis treatment was performed in this study. METHODS: Parameters for rifampicin pharmacokinetics in CSF were estimated using individual-level rifampicin pharmacokinetic data, and the model was externally validated in three separate patient cohorts. Monte Carlo simulations of rifampicin serum and CSF concentrations were performed. The area under the rifampicin CSF concentration-versus-time curve during 24 h (AUC0-24) relative to the minimum inhibitory concentration (MIC) served as the pharmacodynamic target. RESULTS: Across all simulated patients on the first treatment day, 85% attained the target AUC0-24/MIC ratio of 30 under a weight-based dosing scheme approximating 10 mg/kg. At the rifampicin MIC of 0.5 mg/l, the probability of AUC0-24/MIC target attainment was 26%. With an intensified dosing strategy corresponding to 20 mg/kg, target attainment increased to 99%, including 93% with a MIC of 0.5 mg/l. CONCLUSIONS: Under standard dosing guidelines, few TB meningitis patients would be expected to attain therapeutic rifampicin exposures in CSF when the MIC is ≥0.5 mg/l. Either downward adjustment of the rifampicin MIC breakpoint in the context of TB meningitis, or intensified rifampicin dosing upwards of 20 mg/kg/day, would reflect the likelihood of pharmacodynamic target attainment in CSF.


Subject(s)
Antitubercular Agents/cerebrospinal fluid , Rifampin/cerebrospinal fluid , Tuberculosis, Meningeal/cerebrospinal fluid , Adult , Antitubercular Agents/blood , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/therapeutic use , Humans , Microbial Sensitivity Tests , Monte Carlo Method , Rifampin/blood , Rifampin/pharmacokinetics , Tuberculosis, Meningeal/drug therapy
4.
Int J Infect Dis ; 68: 18-23, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29253711

ABSTRACT

OBJECTIVES: Pyrazinamide is a key drug in the first-line treatment regimen for tuberculosis, with a potent sterilizing effect. Although low pyrazinamide peak serum concentrations (Cmax) are associated with poor treatment outcomes, many resource-constrained settings do not have sufficient laboratory capacity to support therapeutic drug monitoring (TDM). The objective of this study was to determine whether a colorimetric test of urine can identify tuberculosis patients with adequate pyrazinamide exposures, as defined by serum Cmax above a target threshold. METHODS: In the derivation study of healthy volunteers, three dose sizes of pyrazinamide were evaluated, and intensive pharmacokinetic blood sampling was performed over an 8-h period, with a timed urine void at 4h post-dosing. Pyrazinamide in urine was isolated by spin column centrifugation with an exchange resin, followed by colorimetric analysis; the absorbance peak at 495nm was measured. The urine assay was then evaluated in a study of 39 HIV/tuberculosis patients in Botswana enrolled in an intensive pharmacokinetic study. Receiver operating characteristics (ROC) curves were used to measure diagnostic accuracy. The guideline-recommended pyrazinamide serum Cmax target of 35mg/l was evaluated in the primary analysis; this target was found to be predictive of favorable outcomes in a clinical study. Following this, a higher serum Cmax target of 58mg/l was evaluated in the secondary analysis. RESULTS: At the optimal cut-off identified in the derivation sample, the urine colorimetric assay was 97% sensitive and 50% specific to identify 35 of 39 HIV/tuberculosis patients with pharmacokinetic target attainment, with an area under the ROC curve of 0.81 (95% confidence interval 0.60-0.97). Diagnostic accuracy was lower at the 58mg/l serum Cmax target, with an area under the ROC curve of 0.68 (95% confidence interval 0.48-0.84). Men were less likely than women to attain either serum pharmacokinetic target. CONCLUSIONS: The urine colorimetric assay was sensitive but not specific for the detection of adequate pyrazinamide pharmacokinetic exposures among HIV/tuberculosis patients in a high-burden setting.


Subject(s)
Colorimetry , Drug Monitoring , Pyrazinamide/therapeutic use , Tuberculosis/drug therapy , Tuberculosis/urine , Adult , Botswana , Cross-Over Studies , Dose-Response Relationship, Drug , Female , HIV Infections/drug therapy , HIV Infections/urine , Humans , Male , Non-Randomized Controlled Trials as Topic , Pyrazinamide/pharmacokinetics , Pyrazinamide/urine , Reproducibility of Results , Sensitivity and Specificity , Treatment Outcome , Young Adult
5.
Tuberc Res Treat ; 2017: 2140974, 2017.
Article in English | MEDLINE | ID: mdl-29430306

ABSTRACT

BACKGROUND: Rifampin malabsorption is frequently observed in tuberculosis patients coinfected with human immunodeficiency virus (HIV) but cannot be predicted by patient factors such as CD4+ T cell count or HIV viral load. METHODS: We sought to describe the relationship between HIV-associated immune activation, measures of gut absorptive capacity and permeability, and rifampin pharmacokinetic parameters in a pilot study of 6 HIV-infected, tuberculosis-uninfected patients who were naïve to antiretroviral therapy. RESULTS: The median rifampin area under the concentration-versus-time curve during the 8-hour observation period was 42.8 mg·hr/L (range: 21.2 to 57.6), with a median peak concentration of 10.1 mg/L (range: 5.3 to 12.5). We observed delayed rifampin absorption, with a time to maximum concentration greater than 2 hours, in 2 of 6 participants. There was a trend towards increased plasma concentrations of sCD14, a marker of monocyte activation in response to bacterial translocation, among participants with delayed rifampin absorption compared to participants with rapid absorption (p = 0.06). CONCLUSIONS: Delayed rifampin absorption may be associated with elevated markers of bacterial translocation among HIV-infected individuals naïve to antiretroviral therapy. This trial is registered with NCT01845298.

6.
J Immunol ; 197(7): 2936-47, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27559046

ABSTRACT

Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.


Subject(s)
Immunoglobulin G/immunology , Receptors, Fc/immunology , Animals , Binding Sites , Genetic Variation/genetics , Humans , Macaca mulatta , Receptors, Fc/genetics , Receptors, Fc/isolation & purification
7.
BMC Infect Dis ; 16: 242, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27250739

ABSTRACT

BACKGROUND: The cost and complexity of current approaches to therapeutic drug monitoring during tuberculosis (TB) therapy limits widespread use in areas of greatest need. We sought to determine whether urine colorimetry could have a novel application as a form of therapeutic drug monitoring during anti-TB therapy. METHODS: Among healthy volunteers, we evaluated 3 dose sizes of rifampin (150 mg, 300 mg, and 600 mg), performed intensive pharmacokinetic sampling, and collected a timed urine void at 4 h post-dosing. The absorbance peak at 475 nm was measured after rifamycin extraction. The optimal cutoff was evaluated in a study of 39 HIV/TB patients undergoing TB treatment in Botswana. RESULTS: In the derivation study, a urine colorimetric assay value of 4.0 × 10(-2) Abs, using a timed void 4 h after dosing, demonstrated a sensitivity of 92 % and specificity of 60 % to detect a peak rifampin concentration (Cmax) under 8 mg/L, with an area under the ROC curve of 0.92. In the validation study, this cutoff was specific (100 %) but insensitive (28 %). We observed similar test characteristics for a target Cmax target of 6.6 mg/L, and a target area under the drug concentration-versus-time curve (AUC0-8) target of 24.1 mg•hour/L. CONCLUSIONS: The urine colorimetric assay was specific but insensitive to detect low rifampin serum concentrations among HIV/TB patients. In future work we will attempt to optimize sampling times and assay performance, with the goal of delivering a method that can translate into a point-of-care assessment of rifampin exposure during anti-TB therapy.


Subject(s)
Antitubercular Agents/therapeutic use , Antitubercular Agents/urine , Drug Monitoring/methods , Rifampin/therapeutic use , Rifampin/urine , Tuberculosis/drug therapy , Urinalysis/methods , Adult , Antitubercular Agents/analysis , Botswana , Colorimetry/methods , Cross-Over Studies , Dose-Response Relationship, Drug , Female , Healthy Volunteers , Humans , Male , ROC Curve , Rifampin/analysis , Sensitivity and Specificity , Specimen Handling , Tuberculosis/urine
8.
Antimicrob Agents Chemother ; 58(12): 7056-71, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25224013

ABSTRACT

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.


Subject(s)
Cervix Uteri/drug effects , HIV-1/drug effects , Phosphorothioate Oligonucleotides/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Reverse Transcription/drug effects , Virus Internalization/drug effects , Animals , Cervix Uteri/virology , Deoxyribose/chemistry , Epithelial Cells/drug effects , Epithelial Cells/virology , Female , Gene Expression , HIV-1/enzymology , HIV-1/genetics , HIV-1/growth & development , Humans , Male , Mice , Mice, Inbred C57BL , Mucous Membrane/drug effects , Mucous Membrane/virology , Organ Culture Techniques , Phosphorothioate Oligonucleotides/chemical synthesis , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Receptors, CXCR4/antagonists & inhibitors , Reverse Transcriptase Inhibitors/chemical synthesis , Sperm Motility/drug effects , Structure-Activity Relationship , Vagina/drug effects , Vagina/virology
9.
Infect Immun ; 82(10): 4080-91, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25024365

ABSTRACT

Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.


Subject(s)
Antibodies, Bacterial/blood , Antitoxins/blood , Bacterial Proteins/immunology , Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Enterotoxins/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Neutralizing/blood , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Cross Protection , Electrophoresis , Enterotoxins/genetics , Female , Injections, Intramuscular , Macaca mulatta , Mice , Mice, Inbred C57BL , Neutralization Tests , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Survival Analysis , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
10.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 4): 520-33, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23519660

ABSTRACT

Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a D-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein-peptide-antibiotic complex. The 2.05 Å resolution MBP-peptide-teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.


Subject(s)
Anti-Bacterial Agents/chemistry , Carrier Proteins/chemistry , Cell Wall/chemistry , Glycopeptides/chemistry , Teicoplanin/chemistry , Anti-Bacterial Agents/metabolism , Carrier Proteins/metabolism , Crystallization , Crystallography, X-Ray , Glycopeptides/metabolism , Ligands , Micromonosporaceae , Protein Binding , Protein Precursors/chemistry , Protein Precursors/metabolism , Teicoplanin/metabolism
11.
ChemMedChem ; 8(3): 426-32, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23361947

ABSTRACT

The development of drug resistance remains a critical problem for current HIV-1 antiviral therapies, creating a need for new inhibitors of HIV-1 replication. We previously reported on a novel anti-HIV-1 compound, N(2)-(phenoxyacetyl)-N-[4-(1-piperidinylcarbonyl)benzyl]glycinamide (14), that binds to the highly conserved phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) binding pocket of the HIV-1 matrix (MA) protein. In this study, we re-evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV-1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-piperidinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)acetamide (18), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV-1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P(2) for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P(2) binding site of MA decreased the antiviral effect of compound 7. Additionally, compound 7 displays a broadly neutralizing anti-HIV activity, with IC(50) values of 7.5-15.6 µM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti-HIV-1 therapeutics.


Subject(s)
Acetanilides/pharmacology , Anti-HIV Agents/pharmacology , HIV Antigens/chemistry , HIV-1/metabolism , Oxadiazoles/pharmacology , Phosphatidylinositol 4,5-Diphosphate/chemistry , Small Molecule Libraries/pharmacology , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency Virus/chemistry , Acetanilides/chemistry , Anti-HIV Agents/chemistry , Binding Sites , Cells, Cultured , HEK293 Cells , HIV Antigens/genetics , HIV Antigens/metabolism , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Molecular Docking Simulation , Oxadiazoles/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Small Molecule Libraries/chemistry , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolism
12.
Bioorg Med Chem Lett ; 23(4): 1132-5, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23305922

ABSTRACT

Due to the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The HIV-1 matrix (MA) protein is an essential viral component with established roles in the assembly of the virus. Using virtual and surface plasmon resonance (SPR)-based screening, we describe the identification of the first small molecule to bind to the HIV-1 MA protein and to possess broad range anti-HIV properties.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , Humans , Models, Molecular , Molecular Targeted Therapy , Surface Plasmon Resonance
13.
Proteins ; 81(2): 271-90, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23011758

ABSTRACT

Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide's affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development.


Subject(s)
Anti-HIV Agents/chemistry , HIV Envelope Protein gp120/chemistry , Peptides/chemistry , Triazoles/chemistry , Anti-HIV Agents/pharmacology , CD4 Antigens/chemistry , CD4 Antigens/metabolism , HIV Envelope Protein gp120/antagonists & inhibitors , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , Humans , Kinetics , Models, Molecular , Mutation/genetics , Peptides/metabolism , Protein Binding , Structure-Activity Relationship , Triazoles/metabolism
14.
J Virol ; 86(16): 8472-81, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22647699

ABSTRACT

The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4'-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity.


Subject(s)
Anti-HIV Agents/pharmacology , Capsid Proteins/antagonists & inhibitors , HIV-1/drug effects , HIV-1/physiology , Virus Uncoating/drug effects , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/toxicity , Calorimetry , Cell Line , Humans , Microbial Sensitivity Tests , Protein Binding , Real-Time Polymerase Chain Reaction , Reverse Transcription/drug effects , Simian Immunodeficiency Virus/drug effects , Surface Plasmon Resonance , Virus Internalization/drug effects , Virus Replication/drug effects
15.
J Med Chem ; 55(7): 3011-20, 2012 Apr 12.
Article in English | MEDLINE | ID: mdl-22380680

ABSTRACT

The homologous recombination (HR) pathway plays a crucial role in the repair of DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs). RAD51, a key protein of HR, possesses a unique activity: DNA strand exchange between homologous DNA sequences. Recently, using a high-throughput screening (HTS), we identified compound 1 (B02), which specifically inhibits the DNA strand exchange activity of human RAD51. Here, we analyzed the mechanism of inhibition and found that 1 disrupts RAD51 binding to DNA. We then examined the effect of 1 on HR and DNA repair in the cell. The results show that 1 inhibits HR and increases cell sensitivity to DNA damage. We propose to use 1 for analysis of cellular functions of RAD51. Because DSB- and ICL-inducing agents are commonly used in anticancer therapy, specific inhibitors of RAD51 may also help to increase killing of cancer cells.


Subject(s)
Antineoplastic Agents/chemistry , Quinazolinones/chemistry , Rad51 Recombinase/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/pharmacology , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA Repair/drug effects , Drug Synergism , HEK293 Cells , Humans , Mice , Mitomycin/pharmacology , Nucleoproteins/metabolism , Protein Binding , Quinazolinones/pharmacology , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombination, Genetic
16.
J Am Chem Soc ; 134(10): 4637-45, 2012 Mar 14.
Article in English | MEDLINE | ID: mdl-22352468

ABSTRACT

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier-target-antibiotic complex. Using native chemical ligation, we have linked the Lys-D-Ala-D-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-D-Ala-D-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.


Subject(s)
Anti-Bacterial Agents/chemistry , Carrier Proteins/chemistry , Teicoplanin/analogs & derivatives , Crystallization , Molecular Structure , Spectrometry, Fluorescence , Surface Plasmon Resonance , Teicoplanin/chemistry
17.
Biochemistry ; 50(14): 2769-79, 2011 Apr 12.
Article in English | MEDLINE | ID: mdl-21417283

ABSTRACT

We sought to identify sequences in the monoclonal antibody m18 complementarity determining regions (CDRs) that are responsible for its interaction with HIV-1 gp120 and inhibition of the envelope receptor binding sites. In the accompanying paper (DOI 10.1021/bi101160r), we reported that m18 inhibits CD4 binding through a nonactivating mechanism that, at the same time, induces conformational effects leading to inhibition of the coreceptor site. Here, we sought to define the structural elements in m18 responsible for these actions. Direct binding and competition analyses using surface plasmon resonance showed that YU-2 gp120 binding is stabilized by a broad paratope of residues in the m18 CDRs. Additionally, several m18 residues were identified for which mutants retained high affinity for gp120 but had suppressed CD4 and 17b inhibition activities. A subset of these mutants did, however, neutralize HXBc2 viral infection. The results obtained in this work demonstrate that the combined m18 paratope contains subsets of residues that are differentially important for the binding and inhibition functions of the m18 neutralizing antibody. The data also add to prior observations that high-affinity antibodies that do not inhibit monomeric gp120 receptor site interactions may still exhibit significant antiviral activity.


Subject(s)
Antibodies, Monoclonal/metabolism , CD4 Antigens/metabolism , Epitopes/metabolism , HIV Envelope Protein gp120/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Binding Sites/drug effects , Binding, Competitive , CD4 Antigens/immunology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/metabolism , Epitopes/chemistry , Epitopes/genetics , HIV Antibodies/chemistry , HIV Antibodies/genetics , HIV Antibodies/metabolism , HIV-1/immunology , HIV-1/metabolism , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation
18.
Biochemistry ; 50(14): 2756-68, 2011 Apr 12.
Article in English | MEDLINE | ID: mdl-21351734

ABSTRACT

We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.


Subject(s)
Antibodies, Neutralizing/metabolism , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , Protein Conformation , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/immunology , Binding Sites/genetics , Binding, Competitive , CD4 Antigens/immunology , CD4 Antigens/metabolism , Calorimetry , Epitopes/immunology , Epitopes/metabolism , HIV Antibodies/immunology , HIV Antibodies/metabolism , HIV Envelope Protein gp120/genetics , HIV-1/immunology , HIV-1/metabolism , Humans , Models, Molecular , Mutation , Protein Binding , Protein Structure, Tertiary , Surface Plasmon Resonance , Thermodynamics
19.
ChemMedChem ; 5(11): 1871-9, 2010 Nov 08.
Article in English | MEDLINE | ID: mdl-20677318

ABSTRACT

In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co-receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy- and amino-terminal residues from the parent 12-residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)-Trp cluster at residues 6 and 7 with ferrocenyl-triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino-terminal residues distal to the central triazole(Pro)-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp120/antagonists & inhibitors , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Peptides/metabolism , Triazoles/metabolism , Amino Acid Sequence , Anti-HIV Agents/chemistry , CD4 Antigens/metabolism , Catalytic Domain , HIV Envelope Protein gp120/chemistry , HIV Infections/drug therapy , HIV-1/metabolism , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Sequence Data , Peptides/chemistry , Surface Plasmon Resonance , Thermodynamics , Triazoles/chemistry
20.
J Mol Recognit ; 22(2): 169-74, 2009.
Article in English | MEDLINE | ID: mdl-18498083

ABSTRACT

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2+3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp120/antagonists & inhibitors , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Organometallic Compounds/chemistry , Peptide Fragments/pharmacology , Triazoles/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Binding Sites , CD4 Antigens/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Ferrous Compounds/chemistry , HIV Antibodies/metabolism , HIV-1/immunology , Humans , Metallocenes , Molecular Mimicry , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Protein Binding , Recombinant Proteins/metabolism , Triazoles/chemical synthesis , Triazoles/chemistry , Virus Internalization/drug effects
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