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1.  Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design 
The Journal of biological chemistry  2004;279(28):29493-29500.
Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible.
PMCID: PMC3491871  PMID: 15102853
enzyme-inhibitor complex; Trypanosoma cruzi; trypanothione reductase; quinacrine mustard; X-ray diffraction
2.  Bis-Acridines as Lead Antiparasitic Agents: Structure-Activity Analysis of a Discrete Compound Library In Vitro▿  
Parasitic diseases are of enormous public health significance in developing countries—a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 Å. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC50) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC50 values against Plasmodium falciparum and Trypanosoma brucei; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent of P. falciparum resistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization.
PMCID: PMC1891397  PMID: 17371810

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