Joseph Sempombe

Mutations in the FMN Domain Modulate MCD Spectra of the Heme Site in the Oxygenase Domain of Inducible Nitric Oxide Synthase

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Spectroscopic characterization of YedY: the role of sulfur coordination in a Mo(V) sulfite oxidase family enzyme form.

Electronic paramagnetic resonance (EPR), electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, Arabidopsis thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from Starkeya novella. Low-energy dithiolene --> Mo and cysteine thiolate --> Mo charge-transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. In particular, a more acute O(oxo)-Mo-S(Cys)-C dihedral angle results in increased cysteine thiolate S --> Mo charge transfer and a large g(1) in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active-site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.

Spectroscopic and Electronic Structure Studies Probing Covalency Contributions to C–H Bond Activation and Transition-State Stabilization in Xanthine Oxidase

A detailed electron paramagnetic resonance (EPR) and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed and serves as a basis for developing a valence-bond description of C-H activation and transition-state (TS) stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde-inhibited XO have been analyzed in order to provide information regarding the relationship between the g, (95,97)Mo hyperfine (A(Mo)), and (13)C hyperfine (A(C)) tensors. Analysis of the EPR spectra has allowed for greater insight into the electronic origin of key delocalizations within the Mo-O(eq)-C fragment and how these contribute to C-H bond activation/cleavage and TS stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo═S π → C-H σ* (ΔE = 24.3 kcal mol(-1)) and C-H σ → Mo═S π* (ΔE = 20.0 kcal mol(-1)) back-donation are important in activating the substrate C-H bond for cleavage. Additional contributions to C-H activation derive from O(eq) lp → C-H σ* (lp = lone pair; ΔE = 8.2 kcal mol(-1)) and S lp → C-H σ* (ΔE = 13.2 kcal mol(-1)) stabilizing interactions. The O(eq)-donor ligand that derives from water is part of the Mo-O(eq)-C fragment probed in the EPR spectra of inhibited XO, and the observation of O(eq) lp → C-H σ* back-donation indicates a key role for O(eq) in activating the substrate C-H bond for cleavage. We also show that the O(eq) donor plays an even more important role in TS stabilization. We find that O(eq) → Mo + C charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal mol(-1)) and the Mo-O(eq)-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-O(eq)-C delocalization at the TS allows for the TS to be described in valence-bond terms as a resonance hybrid of the reactant (R) and product (P) valence-bond wave functions.

Mutation in the flavin mononucleotide domain modulates magnetic circular dichroism spectra of the iNOS ferric cyano complex in a substrate-specific manner.

We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.

Pesticidal Activity of Wild Mushroom Cantharellus cibarius (FR) Extracts against Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) in Stored Maize Grains

Grain damage due to pest infestation is among the top challenges facing cereals production. Maize grains being among the staple food in different parts of the world is prone to destruction by pests such as vertebrates, fungi and insects who are known to affect maize before harvest and during post harvest storage. Sitophilus zeamais is among the potential maize grains infestants. The efforts to control the pest highly depends on the application of synthetic pesticides which are faced by challenges of limited access, fear for toxicity, development of resistance among the pests and environmental pollution. This study aimed at assessing the toxicity, anti-feedant and repellence activities of crude methanol extracts of wild mushroom Cantharellus cibarius on Sitophilus zeamais in stored maize grains towards searching for alternative means of pest control. Assessments were conducted using six levels of concentrations ranging between 0.05 to 0.5 % w/w. Nontreated grains and treated grains with 2% Actellic gold TM 2% dust (0.05% w/w) were used as negative and positive controls respectively. Three replicates were made for each treatment and experiments were conducted in a completely randomized design. The methanol extract at 0.5% w/w concentration demonstrated high toxicity 21 days after treatment killing 66.7% of the pest. Similarly, a 92.5% reduction in grain damage was observed at 0.5% w/w 21 days after treatment compared to nontreated controls. Furthermore, the extracts indicated pest repellence of 98.3% after 24 hours of exposure. The findings render C. cibarius a potential biopesticide for use by subsistence farmers against maize storage pests to support the ongoing Integrated Pests Management strategies. Further studies are recommended on the appropriate frequency and rate of application as well as the maximum duration of protection that can be offered by the extracts.

Development and validation of a high-performance thin-layer chromatographic—densitometric method for the analysis of miconazole in creams

A new high-performance thin-layer chromatographic (HPTLC)—densitometric method which can be employed in the routine analysis of miconazole in creams has been developed and validated as per the International Conference on Harmonization (ICH) guidelines and the United States Pharmacopeia (USP) guidance. The method involves the use of a mobile phase composed of safer reagents for both the user and the environment than the previously available methods. In this method, ethyl acetate—ammonia solution 25% (25:0.5 v/v) was an optimum composition of a solvent system on precoated HPTLC silica gel 60 F254 glass plates with a saturation time of 25 min and migration rate of 10.5 min per 70 mm migration distance. The RF value was 0.57 with no interferences from excipients or solvents at the detection wavelength of 228 nm. The relative standard deviation (RSD) values of 1.14 and 1.98 were obtained for repeatability and intermediate precision, respectively, of the method. The polynomial R2 values computed on three successive days for linearity testing were 0.984, 9.81, and 0.992. Accuracy values between 97.95% and 106.86% were obtained when testing was performed at 80%, 100%, and 120% concentration levels. The method is of acceptable selectivity and reproducibility as well as of easy use.

Formulation development and optimization of Lamivudine 300 mg and Tenofovir Disoproxil Fumarate (TDF) 300 mg FDC tablets by D-optimal mixture design

The usage of fixed dose combination (FDC) tablets of Lamivudine and Tenofovir Disoproxil Fumarate (TDF) is increasing due to increased incidences of HIV/Hepatitis B and HIV/TB co-infections. This is likely to increase the financial crisis due to limited resources for funding procurement of ready-made products from the pharmaceuticals manufacturing leading countries. Therefore, production of local oral tablets containing Lamivudine and TDF FDC is inevitable. Lamivudine 300 mg/TDF 300 mg tablets were developed and optimized by D-optimal mixture design and produced by direct compression technique. Twenty trial formulations with independent variables, including PVP-CL 1–12.00%, PVP-K30 1–10.00%, starch-1500 2.5–12.5% and Avicel-PH102 2–19.25% were prepared by direct compression technique. The formulations were assessed on assay, dissolution, friability, weight variation and disintegration time. It was found that assay ranged from 98.13–101.95% for Lamivudine, 98.25–102.84 for TDF, both were within the in-house assay specification of 95 to 105%. Dissolution at single point was above 80% for Lamivudine 93.96–100.55% and 95.85–103.15% for TDF, disintegration time was between 1.92–66.33 min and friability 0.06–12.56%. Out of twenty formulation trials, eight formulations had all parameters in proven acceptable range. On optimization, one formulation with independent variables, PVP-CL 5.67%, PVP-K30 1.00%, Starch-1500 5.76% was selected. The optimized formulation was comparable to the reference product on the market with similarity factor (f2) and difference factor (f1) within the acceptable range for both Lamivudine and TDF.

Development and Validation of a Thin-Layer Chromatographic-Densitometric Method for the Analysis of Clotrimazole Vaginal Tablets

A thin-layer chromatography (TLC) method for the analysis of clotrimazole was developed and validated according to the International Conference on Harmonization (ICH) and the United States Pharmacopeia (USP) guidelines. The developed method will be used for a qualitative and quantitative analysis of clotrimazole in vaginal tablets. The method was developed using mobile phase containing toluene-acetone (15:10 v/v) on precoated TLC silica gel 60 F254 glass plates at a detection wavelength of 215 nm using reflectance absorbance and saturation time of 20 min. Densitometric analysis showed clotrimazole being retained at RF of 0.27. The method had acceptable level of specificity where no interference was observed between clotrimazole peaks to that of blank. The calibration curve of clotrimazole was estimated using both linear and polynomial regression function in the range of 1000–2400 ng spot−1 with regression coefficient, r2, of 0.992 for linear and 0.999 for polynomial regression. The accuracy at nominal concentration of clotrimazole was found to be 99.14%, and % RSD was 1.13% for repeatability and 1.30% for intermediate precision that involved two analysts. The developed analytical method is useful for the determination of clotrimazole in the finished drug formulation. Also it helps to identify substandard and fake medicines labeled as clotrimazole during postmarketing surveillance of the drug and especially in resource-constrained countries.