Mutation of the Plasmodium falciparum Flavokinase Confers Resistance to Roseoflavin and 8-Aminoriboflavin.

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.

Roseoflavin, a Natural Riboflavin Analogue, Possesses In Vitro and In Vivo Antiplasmodial Activity.

The ability of the human malaria parasite Plasmodium falciparum to access and utilize vital nutrients is critical to its growth and proliferation. Molecules that interfere with these processes could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilization of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focusing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4 to 5 days. Our data are consistent with riboflavin metabolism and/or the utilization of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

Bioactivity Profiles of Cytoprotective Short-Chain Quinones.

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.

Application of Carbon Nanotubes in Chiral and Achiral Separations of Pharmaceuticals, Biologics and Chemicals.

Carbon nanotubes (CNTs) possess unique mechanical, physical, electrical and absorbability properties coupled with their nanometer dimensional scale that renders them extremely valuable for applications in many fields including nanotechnology and chromatographic separation. The aim of this review is to provide an updated overview about the applications of CNTs in chiral and achiral separations of pharmaceuticals, biologics and chemicals. Chiral single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) have been directly applied for the enantioseparation of pharmaceuticals and biologicals by using them as stationary or pseudostationary phases in chromatographic separation techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and gas chromatography (GC). Achiral MWCNTs have been used for achiral separations as efficient sorbent objects in solid-phase extraction techniques of biochemicals and drugs. Achiral SWCNTs have been applied in achiral separation of biological samples. Achiral SWCNTs and MWCNTs have been also successfully used to separate achiral mixtures of pharmaceuticals and chemicals. Collectively, functionalized CNTs have been indirectly applied in separation science by enhancing the enantioseparation of different chiral selectors whereas non-functionalized CNTs have shown efficient capabilities for chiral separations by using techniques such as encapsulation or immobilization in polymer monolithic columns.

Hybrid protein-inorganic nanoparticles: From tumor-targeted drug delivery to cancer imaging.

Recently, a great interest has been paid to the development of hybrid protein-inorganic nanoparticles (NPs) for drug delivery and cancer diagnostics in order to combine the merits of both inorganic and protein nanocarriers. This review primarily discusses the most outstanding advances in the applications of the hybrids of naturally-occurring proteins with iron oxide, gadolinium, gold, silica, calcium phosphate NPs, carbon nanotubes, and quantum dots in drug delivery and cancer imaging. Various strategies that have been utilized for the preparation of protein-functionalized inorganic NPs and the mechanisms involved in the drug loading process are discussed. How can the protein functionalization overcome the limitations of colloidal stability, poor dispersibility and toxicity associated with inorganic NPs is also investigated. Moreover, issues relating to the influence of protein hybridization on the cellular uptake, tumor targeting efficiency, systemic circulation, mucosal penetration and skin permeation of inorganic NPs are highlighted. A special emphasis is devoted to the novel approaches utilizing the protein-inorganic nanohybrids in combined cancer therapy, tumor imaging, and theranostic applications as well as stimuli-responsive drug release from the nanohybrids.