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.

Synthesis and antileishmanial activity of novel pyridinium-hydrazone derivatives

“…Most of them include nitrogen heterocycle rings such as quinolines 9 , quinazolines 10 , acridines 11 , pyrimidines 12 , pyrazols 13 , pyridines 8 , benzothiazoles 14 , imidazoles 15 , thiadiazoles 16 and include functional structures such as alkyl phospholipids 17 , ether phospholipids 18 , chalcones 19 , amidines 20 , oximes 21 , amidoxime 22 , hydrazones 23 and hydrazides 24,25 . On the other hand, a number of quaternized amine derivatives such as thiadiazolium-phenylamine 26,27 , arylisoquinolinium 28 , imidazo-pyridinium 29 , alkylammonium-phenothiazines 30 , indolo-quinolinium 31 and naphthalimide-amonnium 32 were previously reported to exert antiprotozoal properties. Antiprotozoal activity of quaternary ammonium compounds against Acanthamoeba spp.…”

Virtual Screening of Combinatorial Libraries across a Gene Family: in Search of Inhibitors of Giardia lamblia Guanine Phosphoribosyltransferase

“…A number of groups have reported on the successful design of inhibitors directed against trypanosomal (2,4,(15)(16), leishmanial (6), malarial (19), and tritrichomonal (3, 27) targets active in the 10 nM to 50 M range. However, with the number of compounds that could be generated by combinatorial chemistry growing exponentially, it has become apparent that chemical diversity has surpassed the capacity of high-throughput screening.…”

Thiol Metabolism of the Trypanosomatids as Potential Drug Targets

“…Although potent inhibitors of TR could be developed by means of molecular modeling and quantitative docking techniques (22), it was found that several different modes of binding of such compounds to TR are energetically allowed. The authors conclude that with inhibitors not docked to a unique locus, it may be difficult to obtain good X-ray diffraction data for their binding to TR.…”