Evaluation of the feasibility and efficacy of point-of-care antibody tests for biomarker guided management of COVID-19.

BACKGROUND: Biomarker guided therapy could improve management of COVID-19 inpatients. Although some results indicate that antibody tests are prognostic, little is known about patient management using point-of-care (POC) antibody tests. METHODS: COVID-19 inpatients were recruited to evaluate 2 POC tests: LumiraDX and RightSign. Ease of use data was collected. Blood was also collected for centralized testing using established antibody assays (GenScript cPass). A nested case-control study assessed if POC tests conducted on stored specimens were predictive of time to sustained recovery, mortality, and a composite safety outcome. RESULTS: While both POC tests exhibited moderate agreement with the GenScript assay (both agreeing with 89% of antibody determinations), they were significantly different from the GenScript assay. Treating the GenScript assay as the gold standard, the LumiraDX assay had 99.5% sensitivity and 58.1% specificity while the RightSign assay had 89.5% sensitivity and 84.0% specificity. The LumiraDX assay frequently gave indeterminant results. Both tests were significantly associated with clinical outcomes. CONCLUSIONS: Although both POC tests deviated moderately from the GenScript assay, they predicted outcomes of interest. The RightSign test was easier to use and was more likely to detect those lacking antibody compared to the LumiraDX test treating GenScript as the gold standard.

Early trajectories of virological and immunological biomarkers and clinical outcomes in patients admitted to hospital for COVID-19: an international, prospective cohort study.

BACKGROUND: Serial measurement of virological and immunological biomarkers in patients admitted to hospital with COVID-19 can give valuable insight into the pathogenic roles of viral replication and immune dysregulation. We aimed to characterise biomarker trajectories and their associations with clinical outcomes. METHODS: In this international, prospective cohort study, patients admitted to hospital with COVID-19 and enrolled in the Therapeutics for Inpatients with COVID-19 platform trial within the Accelerating COVID-19 Therapeutic Interventions and Vaccines programme between Aug 5, 2020 and Sept 30, 2021 were included. Participants were included from 108 sites in Denmark, Greece, Poland, Singapore, Spain, Switzerland, Uganda, the UK, and the USA, and randomised to placebo or one of four neutralising monoclonal antibodies: bamlanivimab (Aug 5 to Oct 13, 2020), sotrovimab (Dec 16, 2020, to March 1, 2021), amubarvimab-romlusevimab (Dec 16, 2020, to March 1, 2021), and tixagevimab-cilgavimab (Feb 10 to Sept 30, 2021). This trial included an analysis of 2149 participants with plasma nucleocapsid antigen, anti-nucleocapsid antibody, C-reactive protein (CRP), IL-6, and D-dimer measured at baseline and day 1, day 3, and day 5 of enrolment. Day-90 follow-up status was available for 1790 participants. Biomarker trajectories were evaluated for associations with baseline characteristics, a 7-day pulmonary ordinal outcome, 90-day mortality, and 90-day rate of sustained recovery. FINDINGS: The study included 2149 participants. Participant median age was 57 years (IQR 46-68), 1246 (58·0%) of 2149 participants were male and 903 (42·0%) were female; 1792 (83·4%) had at least one comorbidity, and 1764 (82·1%) were unvaccinated. Mortality to day 90 was 172 (8·0%) of 2149 and 189 (8·8%) participants had sustained recovery. A pattern of less favourable trajectories of low anti-nucleocapsid antibody, high plasma nucleocapsid antigen, and high inflammatory markers over the first 5 days was observed for high-risk baseline clinical characteristics or factors related to SARS-CoV-2 infection. For example, participants with chronic kidney disease demonstrated plasma nucleocapsid antigen 424% higher (95% CI 319-559), CRP 174% higher (150-202), IL-6 173% higher (144-208), D-dimer 149% higher (134-165), and anti-nucleocapsid antibody 39% lower (60-18) to day 5 than those without chronic kidney disease. Participants in the highest quartile for plasma nucleocapsid antigen, CRP, and IL-6 at baseline and day 5 had worse clinical outcomes, including 90-day all-cause mortality (plasma nucleocapsid antigen hazard ratio (HR) 4·50 (95% CI 3·29-6·15), CRP HR 3·37 (2·30-4·94), and IL-6 HR 5·67 (4·12-7·80). This risk persisted for plasma nucleocapsid antigen and CRP after adjustment for baseline biomarker values and other baseline factors. INTERPRETATION: Patients admitted to hospital with less favourable 5-day biomarker trajectories had worse prognosis, suggesting that persistent viral burden might drive inflammation in the pathogenesis of COVID-19, identifying patients that might benefit from escalation of antiviral or anti-inflammatory treatment. FUNDING: US National Institutes of Health.

SARS-CoV-2 RNA and Nucleocapsid Antigen Are Blood Biomarkers Associated With Severe Disease Outcomes That Improve in Response to Remdesivir.

BACKGROUND: Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter Adaptive COVID-19 Treatment Trial 1, which randomized patients to remdesivir or placebo. METHODS: Longitudinal specimens collected during hospitalization from a substudy of 642 patients with COVID-19 were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS: Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95% CI, 1.40-2.71) for levels >245 pg/mL vs 1.04 (95% CI, .76-1.42) for levels <245 pg/mL. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS: Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy. CLINICAL TRIAL REGISTRATION: NCT04280705 (ClinicalTrials.gov).

Novel subtypes of severe COVID-19 respiratory failure based on biological heterogeneity: a secondary analysis of a randomized controlled trial.

BACKGROUND: Despite evidence associating inflammatory biomarkers with worse outcomes in hospitalized adults with COVID-19, trials of immunomodulatory therapies have met with mixed results, likely due in part to biological heterogeneity of participants. Latent class analysis (LCA) of clinical and protein biomarker data has identified two subtypes of non-COVID acute respiratory distress syndrome (ARDS) with different clinical outcomes and treatment responses. We studied biological heterogeneity and clinical outcomes in a multi-institutional platform randomized controlled trial of adults with severe COVID-19 hypoxemic respiratory failure (I-SPY COVID). METHODS: Clinical and plasma protein biomarker data were analyzed from 400 trial participants enrolled from September 2020 until October 2021 with severe COVID-19 requiring ≥ 6 L/min supplemental oxygen. Seventeen hypothesis-directed protein biomarkers were measured at enrollment using multiplex Luminex panels or single analyte enzyme linked immunoassay methods (ELISA). Biomarkers and clinical variables were used to test for latent subtypes and longitudinal biomarker changes by subtype were explored. A validated parsimonious model using interleukin-8, bicarbonate, and protein C was used for comparison with non-COVID hyper- and hypo-inflammatory ARDS subtypes. RESULTS: Average participant age was 60 ± 14 years; 67% were male, and 28-day mortality was 25%. At trial enrollment, 85% of participants required high flow oxygen or non-invasive ventilation, and 97% were receiving dexamethasone. Several biomarkers of inflammation (IL-6, IL-8, IL-10, sTNFR-1, TREM-1), epithelial injury (sRAGE), and endothelial injury (Ang-1, thrombomodulin) were associated with 28- and 60-day mortality. Two latent subtypes were identified. Subtype 2 (27% of participants) was characterized by persistent derangements in biomarkers of inflammation, endothelial and epithelial injury, and disordered coagulation and had twice the mortality rate compared with Subtype 1. Only one person was classified as hyper-inflammatory using the previously validated non-COVID ARDS model. CONCLUSIONS: We discovered evidence of two novel biological subtypes of severe COVID-19 with significantly different clinical outcomes. These subtypes differed from previously established hyper- and hypo-inflammatory non-COVID subtypes of ARDS. Biological heterogeneity may explain inconsistent findings from trials of hospitalized patients with COVID-19 and guide treatment approaches.

Effect of Neutralizing Monoclonal Antibody Treatment on Early Trajectories of Virologic and Immunologic Biomarkers in Patients Hospitalized With COVID-19.

BACKGROUND: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood. METHODS: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and D-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models. RESULTS: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P < .001), and a steeper rate of decline through the first 5 days (P < .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale. CONCLUSIONS: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed. CLINICAL TRIALS REGISTRATION: NCT04501978.

Long-term persistence of transcriptionally active 'defective' HIV-1 proviruses: implications for persistent immune activation during antiretroviral therapy.

OBJECTIVES: People with HIV-1 (PWH) on effective antiretroviral therapy (ART) continue to exhibit chronic systemic inflammation, immune activation, and persistent elevations in markers of HIV-1 infection [including HIV-DNA, cell-associated HIV-RNA (CA HIV-RNA), and antibodies to HIV-1 proteins] despite prolonged suppression of plasma HIV-RNA levels less than 50 copies/ml. Here, we investigated the hypothesis that nonreplicating but transcriptionally and translationally competent 'defective' HIV-1 proviruses may be one of drivers of these phenomena. DESIGN: A combined cohort of 23 viremic and virologically suppressed individuals on ART were studied. METHODS: HIV-DNA, CA HIV-RNA, western blot score (measure of anti-HIV-1 antibodies as a surrogate for viral protein expression in vivo ), and key biomarkers of inflammation and coagulation (IL-6, hsCRP, TNF-alpha, tissue factor, and D-dimer) were measured in peripheral blood and analyzed using a combined cross-sectional and longitudinal approaches. Sequences of HIV-DNA and CA HIV-RNA obtained via 5'-LTR-to-3'-LTR PCR and single-genome sequencing were also analyzed. RESULTS: We observed similar long-term persistence of multiple, unique, transcriptionally active 'defective' HIV-1 provirus clones (average: 11 years., range: 4-20 years) and antibody responses against HIV-1 viral proteins among all ART-treated participants evaluated. A direct correlation was observed between the magnitude of HIV-1 western blot score and the levels of transcription of 'defective' HIV-1 proviruses ( r  = 0.73, P  < 0.01). Additional correlations were noted between total CD8 + T-cell counts and HIV-DNA ( r  = 0.52, P  = 0.01) or CA HIV-RNA ( r  = 0.65, P  < 0.01). CONCLUSION: These findings suggest a novel interplay between transcription and translation of 'defective' HIV-1 proviruses and the persistent immune activation seen in the setting of treated chronic HIV-1 infection.

Clinical evaluation of commercial SARS-CoV-2 serological assays in a malaria endemic setting.

The levels of immune response to SARS-CoV-2 infection or vaccination are poorly understood in African populations and is complicated by cross-reactivity to endemic pathogens as well as differences in host responsiveness. To begin to determine the best approach to minimize false positive antibody levels to SARS-CoV-2 in an African population, we evaluated three commercial assays, namely Bio-Rad Platelia SARS-CoV-2 Total Antibody (Platelia), Quanterix Simoa Semi-Quantitative SARS-CoV-2 IgG Antibody Test (anti-Spike), and the GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (cPass) using samples collected in Mali in West Africa prior to the emergence of SARS-CoV-2. A total of one hundred samples were assayed. The samples were categorized in two groups based on the presence or absence of clinical malaria. Overall, thirteen out of one hundred (13/100) samples were false positives with the Bio-Rad Platelia assay and one of the same one hundred (1/100) was a false positive with the anti-Spike IgG Quanterix assay. None of the samples tested with the GenScript cPass assay were positive. False positives were more common in the clinical malaria group, 10/50 (20%) vs. the non-malaria group 3/50 (6%); p = 0.0374 using the Bio-Rad Platelia assay. Association between false positive results and parasitemia by Bio-Rad remained evident, after adjusting for age and sex in multivariate analyses. In summary, the impact of clinical malaria on assay performance appears to depend on the assay and/or antigen being used. A careful evaluation of any given assay in the local context is a prerequisite for reliable serological assessment of anti-SARS-CoV-2 humoral immunity.

Vaccination Ameliorates Cellular Inflammatory Responses in SARS-CoV-2 Breakthrough Infections.

BACKGROUND: Data on cellular immune responses in persons with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection following vaccination are limited. The evaluation of these patients with SARS-CoV-2 breakthrough infections may provide insight into how vaccinations limit the escalation of deleterious host inflammatory responses. METHODS: We conducted a prospective study of peripheral blood cellular immune responses to SARS-CoV-2 infection in 21 vaccinated patients, all with mild disease, and 97 unvaccinated patients stratified based on disease severity. RESULTS: We enrolled 118 persons (aged 50 years [SD 14.5 years], 52 women) with SARS-CoV-2 infection. Compared to unvaccinated patients, vaccinated patients with breakthrough infections had a higher percentage of antigen-presenting monocytes (HLA-DR+), mature monocytes (CD83+), functionally competent T cells (CD127+), and mature neutrophils (CD10+); and lower percentages of activated T cells (CD38+), activated neutrophils (CD64+), and immature B cells (CD127+CD19+). These differences widened with increased disease severity in unvaccinated patients. Longitudinal analysis showed that cellular activation decreased over time but persisted in unvaccinated patients with mild disease at 8-month follow-up. CONCLUSIONS: Patients with SARS-CoV-2 breakthrough infections exhibit cellular immune responses that limit the progression of inflammatory responses and suggest mechanisms by which vaccination limits disease severity. These data may have implications for developing more effective vaccines and therapies. Clinical Trials Registration. NCT04401449.

The Association of Baseline Plasma SARS-CoV-2 Nucleocapsid Antigen Level and Outcomes in Patients Hospitalized With COVID-19.

BACKGROUND: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19. OBJECTIVE: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2. DESIGN: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge. SETTING: 114 centers in 10 countries. PARTICIPANTS: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms. MEASUREMENTS: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively. RESULTS: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity. LIMITATIONS: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available. CONCLUSION: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

Inflammatory Biomarkers Do Not Differ Between Persistently Seronegative vs Seropositive People With HIV After Treatment in Early Acute HIV Infection.

Persistent viral activity may cause enduring seropositivity and inflammation in treated people with HIV (PWH). We compared inflammatory biomarkers between early treated PWH who remained seronegative or seroconverted and found similar levels of D-dimer, soluble cluster of differentiation 14, C-reactive protein, and interleukin-6, indicating that seronegativity does not affect chronic inflammation in early treated PWH.

Long-term treatment with dopamine D3 receptor agonists induces a behavioral switch that can be rescued by blocking the dopamine D1 receptor.

Restless legs syndrome (RLS) is commonly treated with the dopamine agonists pramipexole, and rotigotine, which target the inhibitory dopamine receptor subtype D3R. While initially highly effective, these compounds lose their efficacy in treating RLS over time, and long-term therapy regularly leads to a worsening of the symptoms (augmentation). This dopamine agonist-induced augmentation has become a prime concern in the treatment of RLS, and while alternate therapies are being proposed, the mechanisms leading to augmentation remain opaque. Evidence suggests that the prolonged D3R treatment may lead to a hyper-dopaminergic state and involve the excitatory dopamine D1 receptor (D1R) subtype. We here present an animal model in which we can test acute and long-term effects of dopamine D3R agonists in a behavior setting relevant to RLS and in which we can induce a switch of the drug effect similar to the one observed in RLS patients under chronic therapy. We also present evidence that we can reverse this long-term effect by blocking the D1R. Together, data from our new animal model indicate that the mechanisms leading to augmentation in RLS patients after long-term use of the currently used dopamine receptor agonists may be related to a D3R-induced upregulation of the D1R system. As such, our model can be used to assess the interactions between D3R and D1R and unravel the mechanisms that lead to augmentation, and it has the potential to serve as a Launchpad for the development of new pharmacological strategies for the treatment of both RLS and augmentation.

Morphine responsiveness to thermal pain stimuli is aging-associated and mediated by dopamine D1 and D3 receptor interactions.

Morphine actions involve the dopamine (DA) D1 and D3 receptor systems (D1R and D3R), and the responses to morphine change with age. We here explored in differently aged wild-type (WT) and D3R knockout mice (D3KO) the interactions of the D1R/D3R systems with morphine in vivo at three different times of the animals' lifespan (2months, 1year, and 2years). We found that: (1) thermal pain withdrawal reflexes follow an aging-associated phenotype, with relatively longer latencies at 2months and shorter latencies at 1year, (2) over the same age range, a dysfunction of the D3R subtype decreases reflex latencies more than aging alone, (3) morphine altered reflex responses in a dose-dependent manner in WT animals and changed at its higher dose the phenotype of the D3KO animals from a morphine-resistant state to a morphine-responsive state, (4) block of D1R function had an aging-dependent effect on thermal withdrawal latencies in control animals that, in old animals, was stronger than that of low-dose morphine. Lastly, (5) block of D1R function in young D3KO animals mimicked the behavioral phenotype observed in the aged WT. Our proof-of-concept data from the rodent animal model suggest that, with age, block of D1R function may be considered as an alternative to the use of morphine, to modulate the response to painful stimuli.

Opposing aging-related shift of excitatory dopamine D1 and inhibitory D3 receptor protein expression in striatum and spinal cord.

Normal aging is associated with a decrease in motor function, a concomitant increase in muscle stiffness and tone, and a decrease in dopamine (DA) levels in the spinal cord. The striatum plays a critical role in the control of motor function, and it receives strong DA innervation from the substantia nigra. However, locomotor activity also requires the activation of motoneurons in the lumbar spinal cord, which in the mouse express all five DA receptor subtypes (D1-D5). Of these, the D3 receptor (D3R) expresses the highest affinity to DA and mediates inhibitory actions, while activation of the lower-affinity D1 receptor (D1R) system promotes excitatory effects. To test whether the aging-related decrease in DA levels is associated with corresponding changes in DA receptor protein expression levels, we probed with Western blot and immunohistochemical techniques for D1R and D3R protein expression levels over the normal life span of the mouse. We found that with age D1R expression levels increased in both striatum and spinal cord, while D3R expression levels remained stable in the striatum or slightly decreased in the spinal cord. The resulting D1-to-D3 ratio indicates a strong upregulation of D1R-mediated pathways in old animals, which is particularly pronounced in the lumbar spinal cord. These data suggest that aging may be associated with a shift in DA-mediated pathways in striatum and spinal cord, which in turn could be an underlying factor in the emergence of aging- and DA-related motor dysfunctions such as Parkinson's disease or Restless Legs Syndrome (RLS).

Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity.

EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies.

Transient kinetics of aminoglycoside phosphotransferase(3')-IIIa reveals a potential drug target in the antibiotic resistance mechanism.

Aminoglycoside phosphotransferases are bacterial enzymes responsible for the inactivation of aminoglycoside antibiotics by O-phosphorylation. It is important to understand the mechanism of enzymes in order to find efficient drugs. Using rapid-mixing methods, we studied the transient kinetics of aminoglycoside phosphotransferase(3')-IIIa. We show that an ADP-enzyme complex is the main steady state intermediate. This intermediate interacts strongly with kanamycin A to form an abortive complex that traps the enzyme in an inactive state. A good strategy to prevent the inactivation of aminoglycosides would be to develop uncompetitive inhibitors that interact with this key ADP-enzyme complex.

Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues.

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.

Interaction of human 3-phosphoglycerate kinase with its two substrates: is substrate antagonism a kinetic advantage?

Substrate antagonism has been described for a variety of enzymes with more than one substrate and is characterized by a lowering of the affinity of one substrate in the presence of the other(s). 3-Phosphoglycerate kinase (PGK) catalyzes phosphotransfer from 1,3-bisphosphoglycerate (bPG) to ADP to give 3-phosphoglycerate (PG) and ATP, and is subject to substrate antagonism. Because of the instability of bPG, antagonism has only been described between PG and ATP or ADP. Here, we show that antagonism also occurs between bPG and ADP. Using the stopped-flow method, we show that the dissociation constant for one substrate increases in the presence of the other, and that this decrease in affinity is mainly due to an increase in the dissociation rate constant. As a consequence, there is an increase in the overall interaction kinetics. Interestingly, in the presence of the mirror image of natural d-ADP, l-ADP (a good substrate for PGK), antagonism is absent. Using rapid-quench-flow, we studied the kinetics of ATP formation. The time courses present the following: (1) a lag with l-ADP, but not with d-ADP, the kinetics of which were similar to the interaction kinetics measured by stopped-flow; (2) a burst that is directed by the phosphotransfer; and (3) a steady-state that is rate limited by the release of product kinetics. Structural explanations for these results are proposed by analyzing the crystallographic structure of the fully closed conformation of PGK in complex with l-ADP, PG, and the transition-state analogue AlF(4)(-) compared to previously determined structures.

Direct kinetic evidence that lysine 215 is involved in the phospho-transfer step of human 3-phosphoglycerate kinase.

3-Phosphoglycerate kinase (PGK) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. PGK is a key enzyme in glycolysis; it catalyzes the reversible reaction 1,3-bisphosphoglycerate + ADP <--> 3-phosphoglycerate + ATP. Here we explored the catalytic role in human PGK of the highly conserved Lys 215 that has been proposed to be essential for PGK function by a transient and equilibrium kinetic study with the active site mutant K215A. By the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type PGK and mutant K215A. By the use of a chemical sampling method (rapid quench flow) under multiple and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild-type PGK follows product formation (presumably product release), whereas with the mutant it is the phospho-transfer step itself that is rate-limiting. Mutant K215A has a low inherent phosphotransferase activity, and to explain this, we carried out a molecular modeling study. This suggests that with the mutant the conserved Arg 65 replaces the missing Lys 215 by helping to position the transferable phospho group during the reaction. Molecular dynamics simulations suggest that in the mutant the closed conformation of the enzyme is stabilized by a salt bridge between Asp 218 and Arg 170 rather than Arg 65 in the wild-type PGK.

Differences in the transient kinetics of the binding of D-ADP and its mirror image L-ADP to human 3-phosphoglycerate kinase revealed by the presence of 3-phosphoglycerate.

L-Nucleosides comprise a new class of antiviral and anticancer agents that are converted in vivo by a cascade of kinases to pharmacologically active nucleoside triphosphates. The last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (PGK), an enzyme that has low specificity for nucleoside diphosphate (NDP): NDP + 1,3-bisphosphoglycerate <--> NTP + 3-phosphoglycerate. Here we compared the kinetics of the formation of the complexes of human PGK with d- and its mirror image l-ADP and the effect of 3-phosphoglycerate (PG) on these by exploiting the fluorescence signal of PGK that occurs upon its interaction with nucleotide substrate. Two types of experiment were carried out: equilibrium (estimation of dissociation constants) and stopped-flow (transient kinetics of the interactions). We show that under our experimental conditions (buffer containing 30% methanol, 4 degrees C) PGK binds d- and l-ADP with similar kinetics. However, whereas PG increased the dissociation rate constant for d-ADP by a factor of 8-which is a kinetic explanation for "substrate antagonism"-PG had little effect on this constant for l-ADP. We explain this difference by a molecular modeling study that showed that the beta-phosphates of d- and l-ADP have different orientations when bound to the active site of human PGK. The difference is unexpected because l-ADP is almost as catalytically competent as d-ADP [ Varga, A. et al. (2008) Biochem. Biophys. Res. Commun. 366, 994-1000].

Molecular characterisation and phylogenetic analysis of Chronic bee paralysis virus, a honey bee virus.

The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.