The crystal structure of halofantrine-ferriprotoporphyrin IX and the mechanism of action of arylmethanol antimalarials

The crystal structure of the complex formed between the antimalarial drug halofantrine and ferriprotoporphyrin IX (Fe(III)PPIX) has been determined by single crystal X-ray diffraction. The structure shows that halofantrine coordinates to the Fe(III) center through its alcohol functionality in addition to π-stacking of the phenanthrene ring over the porphyrin. The length of the Fe(III)-O bond is consistent with an alkoxide and not an alcohol coordinating group. The iron porphyrin is five coordinate and monomeric. Changes in the electronic spectrum of Fe(III)PPIX upon addition of halofantrine base in acetonitrile solution are almost identical to those observed upon addition of quinidine free base in the same solvent. This suggests homologous binding. Molecular mechanics modeling of Fe(III)PPIX complexes of quinidine, quinine, 9-epiquinine and 9-epiquinidine based on this homology suggests that the antimalarially active quinidine and quinine can readily adopt conformations that permit formation of an intramolecular salt bridge between the protonated quinuclidine tertiary amino group and unprotonated heme propionate group, while the inactive epimers 9-epiquinidine and 9-epiquinine have to adopt high energy conformations in order to accommodate such salt bridge formation. We propose that salt bridge formation may interrupt formation of the hemozoin precursor dimer formed during the heme detoxification pathway and so account for the strong activity of the two active isomers.     

Iron(III) protoporphyrin IX complexes of the antimalarial Cinchona alkaloids quinine and quinidine

The antimalarial properties of the Cinchona alkaloids quinine and quinidine have been known for decades. Surprisingly, 9-epiquinine and 9-epiquinidine are almost inactive. A lack of definitive structural information has precluded a clear understanding of the relationship between molecular structure and biological activity. In the current study, we have determined by single crystal X-ray diffraction the structures of the complexes formed between quinine and quinidine and iron(III) protoporphyrin IX (Fe(III)PPIX). Coordination of the alkaloid to the Fe(III) center is a key feature of both complexes, and further stability is provided by an intramolecular hydrogen bond formed between a propionate side chain of Fe(III)PPIX and the protonated quinuclidine nitrogen atom of either alkaloid. These interactions are believed to be responsible for inhibiting the incorporation of Fe(III)PPIX into crystalline hemozoin during its in vivo detoxification. It is also possible to rationalize the greater activity of quinidine compared to that of quinine.     

Linear free energy relationships predict coordination and π-stacking interactions of small molecules with ferriprotoporphyrin IX

In order to better understand the interaction of antimalarial compounds with ferriprotoporphyrin IX (Fe(III)PPIX), association constants of pyridines, imidazoles, amines and phenolates with Fe(III)PPIX and protoporphyrin IX (PPIX) have been measured spectrophotometrically in 40% (v/v) aq. DMSO at pH 7.4. The pH independent log association constants for coordination of nitrogen donor ligands exhibit a linear free energy relationship (LFER) with the pK_a of the donor atom. Association through π-stacking interactions (log K_π) with PPIX and Fe(III)PPIX increases with the number of π-electrons in the aromatic ring system. These findings indicate that in the aqueous milieu of the malaria parasite digestive vacuole, coordination to the Fe(III) center of the porphyrin is necessarily very weak, while π-stacking interactions will be much stronger. On the other hand, in environments in which proton competition is absent, coordination will dominate, with the most basic donor atoms forming the strongest complexes with Fe(III)PPIX. The lipid nanospheres within the digestive vacuole which are now known to be the location of conversion of Fe(III)PPIX to hemozoin could possibly be such an environment, making both types of interaction relevant to the design of new hemozoin inhibitors.     

Theoretical analysis of the binding of iron(III) protoporphyrin IX to 4-methoxyacetophenone thiosemicarbazone via DFT-D3, MEP,QTAIM, NCI,ELF, and LOL studies

Thiosemicarbazones display diverse pharmacological properties, including antimalarial activities. Their pharmacological activities have been studied in depth, but little of this research has focused on their antimalarial mode of action. To elucidate this antimalarial mechanism, we investigated the nature of the interactions between iron(III) protoporphyrin IX (Fe(III)PPIX) and the thione–thiol tautomers of 4-methoxyacetophenone thiosemicarbazone (MAPTSC). Dispersion-corrected density functional theory (DFT-D3), the quantum theory of atoms in molecules (QTAIM), the noncovalent interaction (NCI) index, the electron localization function (ELF), the localized orbital locator (LOL), and thermodynamic calculations were employed in this work. Fe(III)PPIX–MAPTSC binding is expected to inhibit hemozoin formation, thereby preventing Fe(III)PPIX detoxification in plasmodia. Preliminary studies geared toward the identification of atomic binding sites in the thione–thiol tautomers of MAPTSC were carried out using molecular electrostatic potential (MEP) maps and conceptual DFT-based local reactivity indices. The thionic sulfur and the ² N-azomethine nitrogen/thiol sulfur of, respectively, the thione and thiol tautomers of MAPTSC were identified as the most favorable nucleophilic sites for electrophilic attack. The negative values of the computed Fe(III)PPIX–MAPTSC binding energies, enthalpies, and Gibbs free energies are indicative of the existence and stability of Fe(III)PPIX–MAPTSC complexes. MAPTSC–Fe(III) coordinate bonds and strong hydrogen bonds (N–H···O) between the NH₂ group in MAPTSC and the C=O group in one propionate side chain of Fe(III)PPIX are crucial to Fe(III)PPIX–MAPTSC binding. QTAIM, NCI, ELF, and LOL analyses revealed a subtle interplay of weak noncovalent interactions dominated by dispersive-like van der Waals interactions between Fe(III)PPIX and MAPTSC that stabilize the Fe(III)PPIX–MAPTSC complexes.     

Sub-nanosecond photolysis studies of Fe protoporphyrin IX solubilized in neat dimethyl sulfoxide

The present study examines the optical properties of the sub-nanosecond photolytic transient of Fe(II)protoporphyrin IX (Fe(II)PPIX) in neat dimethyl sulfoxide (DMSO). Previous nanosecond studies have revealed that photolysis of the (DMSO)₂Fe(II)PPIX complex in neat DMSO results in the formation of a five-coordinate high-spin (DMSO)Fe(II)PPIX complex within ∼100 ns which decayed with a pseudo-first order rate constant of 2 × 10⁶ s⁻¹ (Larsen et al. (1995) [19]). The results presented here demonstrate that the five-coordinate (DMSO)Fe(II)PPIX species is generated in <100 ps and that no significant changes occur in the kinetic difference between 100 ps and ∼100 ns. The 100 ps transient spectrum of the (DMSO)Fe(II)PPIX complex was also constructed from the kinetic difference spectrum and the equilibrium spectrum of the (DMSO)₂Fe(II)PPIX. The 100 ps transient spectrum exhibits a Soret maximum at ∼432 nm close to that of deoxyMb (435 nm, imidazole coordination) consistent with S-bonded DMSO occupying the fifth coordination site. Neither base elimination is detected on time scales down to 100 ps nor is there evidence for transient O-bonded DMSO followed by linkage isomerization to the equilibrium S-bonded form. The unusually slow rate of DMSO recombination is attributed to electrostatic interactions between DMSO and the five-coordinate heme iron as well as intermolecular interactions between solvent molecules in the bulk, as has been previously proposed.     

Interactions of quinoline antimalarials with hematin in solution

Quinoline antimalarial drugs such as chloroquine and related compounds are believed to act by targeting ferriprotoporphyrin IX (Fe(III)PPIX) in the form of hematin (H(2)O/HO-Fe(III)PPIX), its mu-oxo dimer ([Fe(III)PPIX](2)O) or crystalline beta-hematin ([Fe(III)PPIX](2)) in the malaria parasite. Fe(III)PPIX is formed when the parasite digests host hemoglobin during its intraerythrocytic blood stage. This has led to a number of studies on the interaction of Fe(III)PPIX with quinoline antimalarials and related compounds. This article reviews the spectroscopy, thermodynamics and structures of Fe(III)PPIX-quinoline complexes in solution.     

A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites.

The polymerization of hemoglobin-derived ferric-protoporphyrin IX [Fe(III)PPIX] to inert hemozoin (malaria pigment) is a crucial and unique process for intraerythrocytic plasmodia to prevent heme toxicity and thus a good target for new antimalarials. Quinoline drugs, i.e., chloroquine, and non-iron porphyrins have been shown to block polymerization by forming electronic pi-pi interactions with heme monomers. Here, we report the identification of ferrous-protoporphyrin IX [Fe(II)PPIX] as a novel endogenous anti-malarial. Fe(II)PPIX molecules, released from the proteolysis of hemoglobin, are first oxidized and then polymerized to hemozoin. We obtained Fe(II)PPIX on preparative scale by electrochemical reduction of Fe(III)PPIX, and the reaction was monitored by cyclic voltammetry. Polymerization assays at acidic pH were conducted with the resulting Fe(II)PPIX using a spectrophotometric microassay of heme polymerization adapted to anaerobic conditions and the products characterized by infrared spectroscopy. Fe(II)PPIX (a) did not polymerize and (b) produced a dose-dependent inhibition of Fe(III)PPIX polymerization (IC(50) = 0.4 molar equiv). Moreover, Fe(II)PPIX produced by chemical reduction with thiol-containing compounds gave similar results: a dose-dependent inhibition of heme polymerization was observed using either L-cysteine, N-acetylcysteine, or DL-homocysteine, but not with L-cystine. Cyclic voltammetry confirmed that the inhibition of heme polymerization was due to the Fe(II)PPIX molecules generated by the thiol-mediated reduction of Fe(III)PPIX. These results point to Fe(II)PPIX as a potential endogenous antimalarial and to Fe(III)PPIX reduction as a potential new pharmacological target.     

Metalloporphyrins inhibit beta-hematin (hemozoin) formation

Metal-substituted protoporphyrin IXs (Cr(III)PPIX (1), Co(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6), and Sn(IV)PPIX (7)) act as inhibitors to beta-hematin (hemozoin) formation, a critical detoxification biopolymer of malarial parasites. The central metal ion plays a significant role in the efficacy of the metalloprotoporphyrins to inhibit beta-hematin formation. The efficacy of these compounds correlates well with the water exchange rate for the octahedral aqua complexes of the porphyrin's central metal ion. Under these in vitro reaction conditions, metalloporphyrins 5, 6 and 7 are as much as six times more efficacious than the free ligand protoporphyrin IX in preventing beta-hematin formation and four times as efficacious as chloroquine, while metalloporphyrins 3 and 4 are three to four times more effective at preventing beta-hematin formation than the free protoporphyrin IX base. In contrast, the relatively exchange inert metalloporphyrins 1 and 2 are only as efficacious as the free ligand and only two-thirds as effective as chloroquine. Aggregation studies of the heme:MPPIX using UV-Vis and fluorescence spectroscopies are indicative of the formation of pi-pi hetero-metalloporphyrin assemblies. Thus, hemozoin inhibition is likely prevented by the formation of heme:MPPIX complexes through pi-stacking interactions. The ramifications of such hetero-metalloporphyrin assemblies, in the context of the emerging structural picture of hemozoin, are discussed.     

Speciation and structure of ferriprotoporphyrin IX in aqueous solution: spectroscopic and diffusion measurements demonstrate dimerization,but not μ-oxo dimer formation

Changes in epsilon (393) (the Soret band) of aqueous ferriprotoporphyrin IX [Fe(III)PPIX] with concentration indicate that it dimerizes, but does not form higher aggregates. Diffusion measurements support this observation. The diffusion coefficient of aqueous Fe(III)PPIX is half that of the hydrated monomeric dicyano complex. Much of the apparent instability of aqueous Fe(III)PPIX solutions could be attributed to adsorption onto glass and plastic surfaces. However, epsilon (347) was found to be independent of the aggregation state of the porphyrin and was used to correct for the effects of adsorption. The UV-vis spectrum of the aqueous dimer is not consistent with that expected for a mu-oxo dimer and the (1)H NMR spectrum is characteristic of five-coordinate, high-spin Fe(III)PPIX. Magnetic susceptibility measurements using the Evans method showed that there is no antiferromagnetic coupling in the dimer. By contrast, when the mu-oxo dimer is induced in 10% aqueous pyridine, characteristic UV-vis and (1)H NMR spectra of this species are observed and the magnetic moment is consistent with strong antiferromagnetic coupling. We propose a model in which the spontaneously formed aqueous Fe(III)PPIX dimer involves noncovalent interaction of the unligated faces of two five-coordinate H(2)O/HO-Fe(III)PPIX molecules, with the axial H(2)O/OH(-) ligands directed outwards. This arrangement is consistent with the crystal structures of related five-coordinate iron(III) porphyrins and accounts for the observed pH dependence of the dimerization constant and the spectra of the monomer and dimer. Structures for the aqueous dimer are proposed on the basis of molecular dynamics/simulated annealing calculations using a force field previously developed for modeling metalloporphyrins.     

Evaluation of the influence of the internal aqueous solvent structure on electrostatic interactions at the protein-solvent interface by nonlocal continuum electrostatic approach

The dielectric properties of the polar solvent on the protein-solvent interface at small intercharge distances are still poorly explored. To deconvolute this problem and to evaluate the pair-wise electrostatic interaction (PEI) energies of the point charges located at the protein-solvent interface we used a nonlocal (NL) electrostatic approach along with a static NL dielectric response function of water. The influence of the aqueous solvent microstructure (determined by a strong nonelectrostatic correlation effect between water dipoles within the orientational Debye polarization mode) on electrostatic interactions at the interface was studied in our work. It was shown that the PEI energies can be significantly higher than the energies evaluated by the classical (local) consideration, treating water molecules as belonging to the bulk solvent with a high dielectric constant. Our analysis points to the existence of a rather extended, effective low-dielectric interfacial water shell on the protein surface. The main dielectric properties of this shell (effective thickness together with distance- and orientation-dependent dielectric permittivity function) were evaluated. The dramatic role of this shell was demonstrated when estimating the protein association rate constants.     

Chiral resolution of some antimalarial agents by sub- and supercritical fluid chromatography on an (S)-naphthylurea stationary phase

The behavior of mefloquine, halofantrine, enpiroline, quinine, quinidine, chloroquine and primaquine is studied by subcritical fluid chromatography on a (S)-naphthylurea column (250 mm × 4.6 mm ID) with a subcritical mobile phase composed of carbon dioxide, methanol and triethylamine (flow rate of 3 ml/min). Except for primaquine and chloroquine, each enantiomer was separated at a temperature between 40 and 60°C, and at a pressure below 15 MPa. A 98/2, v/v CO₂/methanol 0.1% triethylamine mixture allowed the separation of halofantrine enantiomers while the enantiomers of the more polar metabolite (N-desbutylhalofantrine) were separated with a 80–20 v/v mixture as used for mefloquine, enpiroline, quinine and quinidine. The influence of temperature, pressure and of the nature of the mobile phase is discussed. © 1993 Wiley-Liss, Inc.     

Interaction of chloroquine and its analogues with heme: An isothermal titration calorimetric study

Quinoline-containing drugs such as chloroquine and quinine have had a long and successful history in antimalarial chemotherapy. Identification of ferriprotoporphyrin IX ([Fe(III)PPIX], haematin) as the drug receptors for these antimalarials called for investigations of the binding affinity, mode of interaction, and the conditions affecting the interaction. The parameters obtained are significant in recent times with the emergence of chloroquine resistant strains of the malaria parasites. This has underlined the need to unravel the molecular mechanism of their action so as to meet the requirement of an alternative to the existing antimalarial drugs. The isothermal titration calorimetric studies on the interaction of chloroquine with haematin lead us to propose an altered mode of binding. The initial recognition is ionic in nature mediated by the propionyl group of haematin with the quaternary nitrogen on CQ. This ionic interaction induces a conformational change, such as to favour binding of subsequent CQ molecules. On the contrary, conditions emulating the cytosolic environment (pH 7.4 and 150 mM salt) reveal the hydrophobic force to be the sole contributor driving the interaction. Interaction of a carefully selected panel of quinoline antimalarial drugs with monomeric ferriprotoporphyrin IX has also been investigated at pH 5.6 mimicking the acidic environment prevalent in the food vacuoles of parasite, the center of drug activity, which are consistent with their antimalarial activity.     

Quinoline antimalarials decrease the rate of beta-hematin formation

The strength of inhibition of beta-hematin (synthetic hemozoin or malaria pigment) formation by the quinoline antimalarial drugs chloroquine, amodiaquine, quinidine and quinine has been investigated as a function of incubation time. In the assay used, beta-hematin formation was brought about using 4.5M acetate, pH 4.5 at 60 degrees C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit beta-hematin formation when relatively short incubation times are used, it was found that beta-hematin eventually forms with longer incubation periods (<8h for chloroquine and >8h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC(50) for inhibition of beta-hematin formation increases markedly with increasing incubation times in the case of the 4-aminoquinolines chloroquine and amodiaquine. By contrast, in the presence of the quinoline methanols quinine and quinidine the IC(50) values increase much more slowly. This results in a partial reversal of the order of inhibition strengths at longer incubation times. Scanning electron microscopy indicates that beta-hematin crystals formed in the presence of chloroquine are more uniform in both size and shape than those formed in the absence of the drug, with the external morphology of these crystallites being markedly altered. The findings suggest that these drugs act by decreasing the rate of hemozoin formation, rather than irreversibly blocking its formation. This model can also explain the observation of a sigmoidal dependence of beta-hematin inhibition on drug concentration.     

On the calculation of electrostatic interactions in proteins

In this paper we present a classical treatment of electrostatic interactions in proteins. The protein is treated as a region of low dielectric constant with spherical charges embedded within it, surrounded by an aqueous solvent of high dielectric constant, which may contain a simple electrolyte. The complete analysis includes the effects of solvent screening, polarization forces, and self energies, which are related to solvation energies. Formulae, and sample calculations of forces and energies, are given for the special case of a spherical protein. Our analysis and model calculations point out that any consistent treatment of electrostatic interactions in proteins should account for the following. Solvent polarization is an important factor in the calculation of pairwise electrostatic interactions. Solvent polarization substantially affects both electrostatic energies and forces acting upon charges. No simple expression for the effective dielectric constant, Deff, can generally be valid, since Deff is a sensitive function of position. Solvent screening of pairwise interactions involving dipolar groups is less effective than the screening of charges. In fact for many interactions involving dipoles, solvent screening can be essentially ignored. The self energy of charges makes a large contribution to the total electrostatic energy of a protein. This must be compensated by specific interactions with other groups in the protein. Strategies for applying our analysis to proteins whose structures are known are discussed.     

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.     

Investigation and optimisation of the drying of reduced glutathione by steered molecular dynamics simulation

The drying of reduced glutathione from a series of aqueous–ethanol binary solutions at 300 K (below human body temperature) and 330 K (above human body temperature) was investigated in detail by steered molecular simulation and an umbrella sampling method with the Gromacs software package and Gromos96(53a6) united atomic force field. The results show that electrostatic interactions between glutathione and solvent represent the main resistance to drying. When the aqueous solution was gradually changed to pure ethanol, the energy of electrostatic interaction between glutathione and solvent molecules increased by 445.088 kJ/mol, and the drying potential of mean force (PMF) free energy also fell by 253.040 kJ/mol. However, an increase in temperature from 300 to 330 K in the aqueous solution only results in an increase of 23.013 kJ/mol in electrostatic interaction energy and a decrease of 34.956 kJ/mol in drying PMF free energy. Furthermore, we show that hydrogen bonding is the major form of electrostatic interaction involved, and directly affects the drying of glutathione. Therefore, choosing water-miscible solvents that minimise hydrogen-bond formation with glutathione will enhance its drying rate, and this is likely to be more efficient than increasing the temperature of the process. Thus, a power-saving technology can be used to produce the high bioactivity medicines.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.     

Interactions de la chloroquine avec la ferriprotoporphyrine IX: Étude par résonance magnétique nucléaire

Chloroquine is still the antimalarial drug which is the most utilized. Nevertheless the molecular mode of action of this drug is not very well understood. When mouse erythrocytes injected with Plasmodium berghei are exposed to chloroquine, the first biochemical event is rapid accumulation of the drug. This process is energy dependent, saturable and competively inhibited by drugs of the same therapeutic class (Quinine, Amodiaquine, Mefloquine). Receptors for chloroquine have been proposed for the process of accumulation. The nature of the chloroquine receptor is presently the subject of debates. The latest hypothesis proposed by Chou and coll. [12], is that ferriprotoporphyrin IX, formed by the degradation of hemoglobin by the parasite, binds to chloroquine with a dissociation constant of 3.5.10⁻⁹ M. We studied here the molecular interactions between these two species by Proton Nuclear Magnetic Resonance in order to elucidate the nature and the geometry of were undertaken.The perturbations of the NMR spectra of chloroquine (10⁻² M) induced by addition of hematin or hemin were measured. Two types of measures were indertaken.The first study carried out in organic solvent (DMSO) has shown that the interaction occured between the acidic functions of hemin and the sidechain nitrogen of chloroquine. The iron atom was not implicated in this process.The second study carried out in aqueous medium (phosphate buffer; 0.1 M; pH = 7) allowed us to demonstrate that chloroquine is able to intercalate into a polymer of hematin. The quinoleic nucleus of chloroquine was intercalated between two dimers of hematin as shown by the broadening of the signal of the quinoleic protons due to very large increase in the correlation time.Finally it was shown that chloroquine is associated as a dimer in aqueous medium by hydrophobic interactions. The association constant is 5.5 M⁻¹.     

Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum remains controversial. By investigating the kinetics of chloroquine accumulation under varying-trans conditions, we recently presented evidence for a saturable and energy-dependent chloroquine efflux system present in chloroquine resistant P. falciparum strains. Here, we further characterize the putative chloroquine efflux system by investigating its substrate specificity using a broad range of different antimalarial drugs. Our data show that preloading cells with amodiaquine, primaquine, quinacrine, quinine, and quinidine stimulates labeled chloroquine accumulation under varying-trans conditions, while mefloquine, halofantrine, artemisinin, and pyrimethamine do not induce this effect. In the reverse of the varying-trans procedure, we show that preloaded cold chloroquine can stimulate quinine accumulation. On the basis of these findings, we propose that the putative chloroquine efflux system is capable of transporting, in addition to chloroquine, structurally related quinoline and methoxyacridine antimalarial drugs. Verapamil and the calcium/calmodulin antagonist W7 abrogate stimulated chloroquine accumulation and energy-dependent chloroquine extrusion. Our data are consistent with a substrate specific and inhibitible drug efflux system being present in chloroquine resistant P. falciparum strains.     

An evaluation of Poisson-Boltzmann electrostatic free energy calculations through comparison with experimental mutagenesis data

For systems involving highly and oppositely charged proteins, electrostatic forces dominate association and contribute to biomolecular complex stability. Using experimental or theoretical alanine-scanning mutagenesis, it is possible to elucidate the contribution of individual ionizable amino acids to protein association. We evaluated our electrostatic free energy calculations by comparing calculated and experimental data for alanine mutants of five protein complexes. We calculated Poisson-Boltzmann electrostatic free energies based on a thermodynamic cycle, which incorporates association in a reference (Coulombic) and solvated (solution) state, as well as solvation effects. We observe that Coulombic and solvation free energy values correlate with experimental data in highly and oppositely charged systems, but not in systems comprised of similarly charged proteins. We also observe that correlation between solution and experimental free energies is dependent on dielectric coefficient selection for the protein interior. Free energy correlations improve as protein dielectric coefficient increases, suggesting that the protein interior experiences moderate dielectric screening, despite being shielded from solvent. We propose that higher dielectric coefficients may be necessary to more accurately predict protein-protein association. Additionally, our data suggest that Coulombic potential calculations alone may be sufficient to predict relative binding of protein mutants.