Role of specific residues in coenzyme binding, charge-transfer complex formation, and catalysis in Anabaena ferredoxin NADP(+)-reductase

AbstractTwo transient charge–transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNRox-NADPH (CTC-1), and FNRrd-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes

Development of Potent and Selective PfCLK3 Inhibitors Based on GSK-TCMDC151 as a New Class of Antimalarials

The kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic, transmission blocking and curative potential. Herein we describe the synthesis of our initial hit TCMDC-135051 1and efforts to establish a SAR with a 7-azaindole-based series. A total of 14 analogues were assessed in a TR-FRET assay against the full recombinant protein kinase PfCLK3 and 10 were further assessed in parasites 3D7 (chloroquine sensitive) strains of P. falciparum. SAR relating to rings A and B was established. These data suggest that TCMDC-135051 1is a promising lead compound for the development of new antimalarials with a novel mechanism of action targeting PfCLK3.</p

Dynamics of the active site architecture in plant-type ferredoxin-NADP + reductases catalytic complexes

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP+ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP+ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.Fil: Sanchez Azqueta, Ana. Universidad de Zaragoza; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Catalano Dupuy, Daniela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lopez Rivero, Arleth Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Tondo, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Medina, Milagros. Universidad de Zaragoza; España. Consejo Superior de Investigaciones Científicas; Españ

Development of Potent PfCLK3 Inhibitors Based on TCMDC-135051 as a New Class of Antimalarials.

The protein kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic, transmission blocking, and curative potential. Herein, we describe the synthesis of our initial hit TCMDC-135051 (1) and efforts to establish a structure-activity relationship with a 7-azaindole-based series. A total of 14 analogues were assessed in a time-resolved fluorescence energy transfer assay against the full-length recombinant protein kinase PfCLK3, and 11 analogues were further assessed in asexual 3D7 (chloroquine-sensitive) strains of P. falciparum parasites. SAR relating to rings A and B was established. These data together with analysis of activity against parasites collected from patients in the field suggest that TCMDC-135051 (1) is a promising lead compound for the development of new antimalarials with a novel mechanism of action targeting PfCLK3

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the; Plasmodium falciparum; protein kinase; Pf; CLK3, which we used in combination with chemogenetics to validate; Pf; CLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for; Pf; CLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of; Pf; CLK3 mediated rapid killing of asexual liver- and blood-stage; P. falciparum; and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against; P. berghei; and; P. knowlesi; Hence, our data establish; Pf; CLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria

Influence of the timing of cardiac surgery on the outcome of patients with infective endocarditis and stroke.

BACKGROUND: The timing of cardiac surgery after stroke in infective endocarditis (IE) remains controversial. We examined the relationship between the timing of surgery after stroke and the incidence of in-hospital and 1-year mortalities. METHODS: Data were obtained from the International Collaboration on Endocarditis-Prospective Cohort Study of 4794 patients with definite IE who were admitted to 64 centers from June 2000 through December 2006. Multivariate logistic regression and Cox regression analyses were performed to estimate the impact of early surgery on hospital and 1-year mortality after adjustments for other significant covariates. RESULTS: Of the 857 patients with IE complicated by ischemic stroke syndromes, 198 who underwent valve replacement surgery poststroke were available for analysis. Overall, 58 (29.3%) patients underwent early surgical treatment vs 140 (70.7%) patients who underwent late surgical treatment. After adjustment for other risk factors, early surgery was not significantly associated with increased in-hospital mortality rates (odds ratio, 2.308; 95% confidence interval [CI], .942-5.652). Overall, probability of death after 1-year follow-up did not differ between 2 treatment groups (27.1% in early surgery and 19.2% in late surgery group, P = .328; adjusted hazard ratio, 1.138; 95% CI, .802-1.650). CONCLUSIONS: There is no apparent survival benefit in delaying surgery when indicated in IE patients after ischemic stroke. Further observational analyses that include detailed pre- and postoperative clinical neurologic findings and advanced imaging data (eg, ischemic stroke size), may allow for more refined recommendations on the optimal timing of valvular surgery in patients with IE and recent stroke syndromes

Impact of early valve surgery on outcome of staphylococcus aureus prosthetic valve infective endocarditis: Analysis in the international collaboration of endocarditis-prospective cohort study

Background. The impact of early valve surgery (EVS) on the outcome of Staphylococcus aureus (SA) prosthetic valve infective endocarditis (PVIE) is unresolved. The objective of this study was to evaluate the association between EVS, performed within the first 60 days of hospitalization, and outcome of SA PVIE within the International Collaboration on Endocarditis-Prospective Cohort Study. Methods. Participants were enrolled between June 2000 and December 2006. Cox proportional hazards modeling that included surgery as a time-dependent covariate and propensity adjustment for likelihood to receive cardiac surgery was used to evaluate the impact of EVS and 1-year all-cause mortality on patients with definite left-sided S. aureus PVIE and no history of injection drug use. Results. EVS was performed in 74 of the 168 (44.3%) patients. One-year mortality was significantly higher among patients with S. aureus PVIE than in patients with non-S. aureus PVIE (48.2% vs 32.9%; P = .003). Staphylococcus aureus PVIE patients who underwent EVS had a significantly lower 1-year mortality rate (33.8% vs 59.1%; P = .001). In multivariate, propensity-adjusted models, EVS was not associated with 1-year mortality (risk ratio, 0.67 [95% confidence interval, .39-1.15]; P = .15). Conclusions. In this prospective, multinational cohort of patients with S. aureus PVIE, EVS was not associated with reduced 1-year mortality. The decision to pursue EVS should be individualized for each patient, based upon infection-specific characteristics rather than solely upon the microbiology of the infection causing PVIE

HACEK infective endocarditis: characteristics and outcomes from a large, multi-national cohort.

The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p&lt;0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p&lt;0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34-9.65; p&lt;0.01) and younger age (OR 0.62; CI 0.49-0.90; p&lt;0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p&lt;0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences

Impact of early valve surgery on outcome of staphylococcus aureus prosthetic valve infective endocarditis: Analysis in the international collaboration of endocarditis-prospective cohort study

Background. The impact of early valve surgery (EVS) on the outcome of Staphylococcus aureus (SA) prosthetic valve infective endocarditis (PVIE) is unresolved. The objective of this study was to evaluate the association between EVS, performed within the first 60 days of hospitalization, and outcome of SA PVIE within the International Collaboration on Endocarditis-Prospective Cohort Study. Methods. Participants were enrolled between June 2000 and December 2006. Cox proportional hazards modeling that included surgery as a time-dependent covariate and propensity adjustment for likelihood to receive cardiac surgery was used to evaluate the impact of EVS and 1-year all-cause mortality on patients with definite left-sided S. aureus PVIE and no history of injection drug use. Results. EVS was performed in 74 of the 168 (44.3%) patients. One-year mortality was significantly higher among patients with S. aureus PVIE than in patients with non-S. aureus PVIE (48.2% vs 32.9%; P = .003). Staphylococcus aureus PVIE patients who underwent EVS had a significantly lower 1-year mortality rate (33.8% vs 59.1%; P = .001). In multivariate, propensity-adjusted models, EVS was not associated with 1-year mortality (risk ratio, 0.67 [95% confidence interval, .39-1.15]; P = .15). Conclusions. In this prospective, multinational cohort of patients with S. aureus PVIE, EVS was not associated with reduced 1-year mortality. The decision to pursue EVS should be individualized for each patient, based upon infection-specific characteristics rather than solely upon the microbiology of the infection causing PVIE