Biochemical and biophysical characterisation of Anopheles gambiae NADPH-cytochrome P450 reductase

As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future

Biochemical Comparison of Anopheles gambiae and Human NADPH P450 Reductases Reveals Different 2′-5′-ADP and FMN Binding Traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (kcat) of 105 s−1 and 88 s−1, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC50 value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (Kd = 410±18 nM) was ∼10 fold weaker than human CPR (Kd = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (Kd = 83±2.0 nM) than the equivalent human domain (Kd = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC50 = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR

Abstracts from the NIHR INVOLVE Conference 2017

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Biochemical and biophysical characterisation of Anopheles gambiae NADPH-cytochrome P450 reductase

As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

High incidence of human brucellosis in a rural Pastoralist community in Kenya, 2015.

BackgroundBrucellosis occurs globally with highly variable incidence in humans from very low in North America and Western Europe to high in the Middle East and Asia. There are few data in Sub-Saharan Africa. This study estimated the incidence of human brucellosis in a pastoralist community in Kenya.MethodsBetween February 2015 and January 2016, we enrolled persons living in randomly selected households in Kajiado County. Free health care was offered at three facilities in the study area. Those who met the study clinical case definition completed a standardized questionnaire on demographics, clinical history and presentation. A blood sample was collected and tested by Rose Bengal test (RBT), then later tested at the Kenya Medical Research Institute laboratory for Brucella IgG and IgM by ELISA. Those who tested positive by both RBT and ELISA (IgG or IgM antibodies) were classified as confirmed while those that only tested positive for IgG or IgM antibodies were classified as probable. Further, sera were tested by polymerase chain reaction using a TaqMan Array Card (TAC) for a panel of pathogens causing AFI including Brucella spp. Annual incidence of brucellosis was calculated as the number of confirmed cases in one year/total number in the study population.ResultsWe enrolled a cohort of 4746 persons in 804 households. Over half (52.3%) were males and the median age was 18 years (Interquartile range (IQR) 9 months- 32 years). A total of 236 patients were enrolled at three health facilities; 64% were females and the median age was 40.5 years (IQR 28-53 years). Thirty-nine (16.5%) were positive for Brucella antibodies by IgG ELISA, 5/236 (2.1%) by IgM ELISA and 4/236 (1.7%) by RBT. Ten percent (22/217) were positive by TAC. We confirmed four (1.7%) brucellosis cases giving an annual incidence of 84/100,000 persons/year (95% CI 82, 87). The incidence did not significantly vary by gender, age and location of residence.ConclusionWe report a high incidence of brucellosis in humans among members of this pastoralist community. Brucellosis was the most common cause of febrile illness in this community