title: Use of an additional hydrophobic binding site, the Z site, in the rational drug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines.
abstract: Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a, 10a-dihydrophenothiazin-10-yl)propyl]dimethylammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine omega-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-L-cysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as >/=5.6 kcal.mol(-1) by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving K(i) values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl](3, 4-dichlorobenzyl)dimethylammonium derivative (K(i) 0.12 microM), was approximately 2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at <1 microM. Antiparasite activity was not solely determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-quaternized chlorpromazime had ED(50) < 1 microM). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED(50) < 1 microM) growth of the amastigote stage of T. cruzi.
authors.affiliation: School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, U.K.
authors.forename: M O
authors.initials: MO
authors.lastname: Khan
authors.forename: S E
authors.initials: SE
authors.lastname: Austin
authors.forename: C
authors.initials: C
authors.lastname: Chan
authors.forename: H
authors.initials: H
authors.lastname: Yin
authors.forename: D
authors.initials: D
authors.lastname: Marks
authors.forename: S N
authors.initials: SN
authors.lastname: Vaghjiani
authors.forename: H
authors.initials: H
authors.lastname: Kendrick
authors.forename: V
authors.initials: V
authors.lastname: Yardley
authors.forename: S L
authors.initials: SL
authors.lastname: Croft
authors.forename: K T
authors.initials: KT
authors.lastname: Douglas
mesh_terms: D000818:Animals; D000977:Antiparasitic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002746:Chlorpromazine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D007893:Leishmania donovani / Q000187:drug effects; D008024:Ligands; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D010640:Phenothiazines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000187:drug effects; D014349:Trypanosoma cruzi / Q000737:chemistry
chemical_list: D000977:Antiparasitic Agents; D004791:Enzyme Inhibitors; D008024:Ligands; D010640:Phenothiazines; D000644:Quaternary Ammonium Compounds; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D002746:Chlorpromazine