Molecular Structures and Solvation of Free Monomeric and Dimeric Ferriheme in Aqueous Solution: Insights from Molecular Dynamics Simulations and Extended X‑ray Absorption Fine Structure Spectroscopy

CHARMM force field parameters have been developed to model nonprotein bound five-coordinate ferriheme (ferriprotoporphyrin IX) species in aqueous solution. Structures and solvation were determined from molecular dynamics (MD) simulations at 298 K of monomeric [HO-ferriheme]^2–, [H₂O-ferriheme]⁻, and [H₂O-ferriheme]⁰; π–π dimeric [(HO-ferriheme)₂]^4–, [(H₂O-ferriheme)­(HO-ferriheme)]^3–, [(H₂O-ferriheme)₂]^2–, and [(H₂O-ferriheme)₂]⁰; and μ-oxo dimeric [μ-(ferriheme)₂O]^4–. Solvation of monomeric species predominated around the axial ligand, meso-hydrogen atoms of the porphyrin ring (H_meso), and the unligated face. Existence of π–π ferriheme dimers in aqueous solution was supported by MD calculations where such dimers remained associated over the course of the simulation. Porphyrin rings were essentially coplanar. In these dimers major and minor solvation was observed around the axial ligand and H_meso positions, respectively. In μ-oxo ferriheme, strong solvation of the unligated face and bridging oxide ligand was observed. The solution structure of the μ-oxo dimer was investigated using extended X-ray absorption fine structure (EXAFS) spectroscopy. The EXAFS spectrum obtained from frozen solution was markedly different from that recorded on dried μ-oxo ferriheme solid. Inclusion of five solvent molecules obtained from spatial distribution functions in the structure generated from MD simulation was required to produce acceptable fits to the EXAFS spectra of the dimer in solution, while the solid was suitably fitted using the crystal structure of μ-oxo ferriheme dimethyl ester which included no solvent molecules

The Effects of Quinoline and Non-Quinoline Inhibitors on the Kinetics of Lipid-Mediated β‑Hematin Crystallization

The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-<i>rac</i>-glycerol was reduced from 0.17 ± 0.04 min^–1 previously measured in Falcon tubes to 0.019 ± 0.002 min^–1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically relevant antimalarial drugs (chloroquine, amodiaquine, quinidine, and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (<i>K</i>_ads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically significant linear correlations between lipid-mediated β-hematin inhibitory activity and <i>K</i>_ads values for quinoline (<i>r</i>² = 0.76, <i>P</i>-value = 0.0046) and non-quinoline compounds (<i>r</i>² = 0.99, <i>P</i>-stat = 0.0006), as well as between parasite inhibitory activity (D10) and <i>K</i>_ads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (<i>r</i>² = 0.64, <i>P</i>-value = 0.0098), provide a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to preformed β-hematin