Antiparasite drug design
(T[-s]2) Rational drug design against parasitic diseases
For some time now Ken Douglas’ laboratory has been involved in using the techniques of rational drug design to introduce structures which could serve as leads for drug design and development in this field. The main activity has been in the diseases caused by trypanosomes and leishmanias and in overcoming resistance to drugs used against malaria.
Publications
For reviews of the lab’s activities in the parasitology area see:
- Rational Drug Design in Parasitology. Parasitology Today (1994) 10, 389-392
- Rational Drug Design using Trypanothione Reductase as a Target for Anti-trypanosomal and Anti-leishmanial Drug leads. Drug Design Discovery (1999) 16, 5-23
Trypanosomal and leishmanial diseases
Major Third World parasitic diseases, including African Sleeping Sickness, Chagas’ disease and leishmaniasis are caused by pathogenic parasites belonging to the order Kinetoplastida, viz., Trypanosoma spp. and Leishmania spp. Infection by these agents can be severely debilitating or even fatal. Currently available drugs are few in number, inadequate in terms of efficacy and often toxic. Moreover, resistance against several of them has already been reported. The second stage of African Sleeping Sickness, in which the trypanosome has invaded the central nervous system, is refractory to attempted treatment by the drugs suramin and pentamidine. The treatment currently available, melarsoprol, is a trivalent arsenical and as such it is no surprise to find that the treatment itself is toxic, leading to deaths in 4-8 % of patients treated with it. Against Chagas’ disease, prevalent in South America (caused by T.cruzi), the two major drugs, nifurtimox and benznidazole, cause considerable concern in terms of their effectiveness and safety. The main drugs in use against Leishmania species are pentavalent antimonials (sodium stibogluconate and meglumine antimoniate) and, as well as toxicity problems, resistance is now being reported. Pentamidine has also been used to treat this set of diseases but the long courses required emphasise the toxicity problems.
It became reasonable to try to harness the power of rational drug design in this area when a fundamental metabolic difference between mammalian host and trypanosomal or leishmanial parasite was discovered, the trypanothione system for redox defence (A.H.Fairlamb et al., Science (1985) 227,1485). In mammals, potential redox damage meets the glutathione (GSH)-based system as a first defence, during the course of which glutathione disulfide (GSSG) is formed, equation (1). Regeneration of protective GSH from GSSG is catalysed by glutathione reductase (GR).
2GSH = GSSG (1)
In trypanosomes and leishmanias an analogous system has evolved, based on trypanothione (T[-s]2), which as the disulfide (T[S]2) differs from GSSG only by the presence of a spermidine cross-link between the two glycyl carboxyl groups (compare GSSG and T[S]2 Figure TSSTUSGSSG). The enzyme trypanothione reductase (TR) reduces T[S]2 to the dithiol form in a manner analogous to GR.
With this discovery of a fundamental metabolic difference, absolute in terms of structural biochemistry, it was soon proposed that TR might form the target for the rational design of anti-trypanosomal drugs. There is an excellent account of the trypanothione system (A. H. Fairlamb, A. Cerami, Annu. Rev. Microbiol. (1992) 46, 695). At a simple level, there is an absolute biochemical difference between host and parasite. Trypanosomes do not contain GR, but rather an analogous enzyme, TR. POWER SL2 Parasite TR differs from host GR in not processing GSSG. Conversely, host GR does not reduce T[S]2. Such mutually exclusive recognition and rejection of cognate substrates between host and parasite argued strongly that selective inhibitor design should be possible. Efficient selective blockade of TR would be expected to compromise the redox defenses of the parasites, increasing their sensitivity to redox-damage based drugs, such as nifurtimox. Thus a TR inhibitor might be expected to be drug in its own right or for co-administration with a redox-active drug such as nifurtimox.
Rational drug design using trypanothione reductase as a target for anti-trypanosomal and anti-leishmanial drug leads
The rational drug design approach was used to discover tricyclic neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for trypanothione reductase over host glutathione reductase. From a homology-modelled structure for trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the enzyme from Crithidia fasciculata, inhibitors based on tricyclic structures POWER SL4 were designed and synthesized (reported in T.J.Benson, J.H. McKie, J.Garforth, A. Borges, A.H.Fairlamb & K.T.Douglas, Biochem. J. (1992) 330, 9). The phenothiazines were reversible inhibitors of trypanothione reductase from Trypanosoma cruzi, linearly competitive with trypanothione as substrate and non-competitive with NADPH, consistent with Ping Pong Bi Bi kinetics. Analogues, prepared to define structure:activity relationships for the active-site, included N-acylpromazines, 2-substituted phenothiazines and tri-substituted promazines (C. Chan, H. Yin, J. Garforth, J. H. McKie, R. Jaouhari, P. Speers, K. T. Douglas, P. J. Rock, V. Yardley, S. L. Croft and A. H. Fairlamb. J. Med. Chem., (1998) 41, 148). Analysis of Ki and I50 inhibition data, using calculated log P and molar refractivity values, gave evidence of a favoured fit of small 2-substituents (especially 2-chloro and 2-trifluoromethyl), with a remote hydrophobic patch on the enzyme accessible for larger, hydrophobic 2-substituents. Inhibition kinetic data also indicated that the phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Selected compounds were tested for in vitro activity against Trypanosoma brucei, T. cruzi and Leishmania donovani, with selective activities in the micromolar range being determined for several. This is reviewed in S. E. Austin, M. O. F. Khan and K. T. Douglas Drug Design Discovery (1999) 16, 5-23.
By using the additional hydrophobic binding site identified above, the Z Site, a new class of stronger inhibitor, quaternary alkylammonium phenothiazines, became possible (M. O. F.Khan, S. E. Austin, C. Chan, H. Yin, D. Marks, S. N. Vaghjiani, H. Kendrick, V. Yardley, S. L. Croft and K. T. Douglas. J. Med. Chem. (2000) 43, 3148).POWER SL6. Substituted-benzyl [3-(2-chloro-4a,10a-dihydro-phenothiazin-10-yl)-propyl]-dimethylammonium salts were competitive inhibitors. 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 analog with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving Ki values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydro-phenothiazin-10-yl)-propyl]-(3,4-dichloro-benzyl)-dimethylammonium derivative, (Ki 0.12 uM), was ~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 mM. 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 ED50 <1 uM). The p-t-butylbenzyl-quaternized analog very strongly inhibited (ED50 <1 uM) growth of the amastigote stage of T. cruzi.
Specific peptide inhibitors of trypanothione reductase with backbone structures unrelated to that of substrate have also been design and tested to provide new lead frameworks (J. H. McKie, J. Garforth, R. Jaouhari, C. Chan, H. Yin, T. Besheya, A.H. Fairlamb and K. T. Douglas. Amino Acids (2001) 20, 145-153. The important proviso here is that the amino-acid structure of the natural substrate is not the basis of inhibitor analogue design. POWER SL3. The inhibitors showed reversible, linear competitive inhibition and the strongest peptide inhibitor to date was found to be N-benzyloxycarbonyl-Ala -Arg-Arg-4-methoxy-b-naphthylamide with a Ki value of 2.4 uM and a selectivity for parasitic enzyme (trypanothione reductase) over the host enzyme (human glutathione reductase) of over 3 orders of magnitude.
Peptoids are molecules which have been developed over the past decade or so to have many of the chemical functionalities of peptides but have distinct structures. The most common family of peptoids uses analogues in which the side-chain normally found on the a–carbon of natural amino acids is re-located so that it effectively lies on the nitrogen atom POWER SL12. A novel lead inhibitor of trypanothione reductase was based on a peptoid structure based on the known strong inhibition by N-benzoyl-Leu-Arg-Arg-b-naphthylamide and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-b-naphthylamide (C. Chan, H. Yin, J.H. McKie, A.H. Fairlamb and K.T., Douglas, Amino Acids (2002) 22, 297). In the target peptoid POWER SL11 the arginyl residues were replaced by alkylimidazolium units and the benzyloxycarbonyl group by the benzylaminocarbonyl function. The peptoid was synthesised using t-butoxycarbonyl protection chemistry and couplings were activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The resulting peptoid was shown to be a competitive inhibitor of recombinant trypanothione reductase from Trypanosoma cruzi with a Ki value of 179 mM and with only weak inhibition of human erythrocyte glutathione reductase (the inhibition of glutathione reductase was at least 291-fold weaker than of trypanothione reductase).
Malaria
With very few safe, effective and cheap antimalarial drugs, the problem of parasite resistance has enormous economic and social consequences, particularly in Africa, where malaria causes ca. 2 million deaths per year and very high levels of morbidity. As a result, ways are being sought to impede the onset and spread of drug resistance and , of course, to try to discover new drugs against malaria.
Pyrimethamine has been employed for many years against malaria, initially on its own, but subsequently in combinations such as Maloprim (pyrimethamine-dapsone) and Fansidar (pyrimethamine-sulfadoxine), the latter a widely used formulation for the treatment of chloroquine-resistant malaria. Pyronaridine, a new Chinese drug, has been tested against malaria in combination with pyrimethamine. Pyrimethamine has also been used recently against toxoplasmic encephalitis relapses and Pneumocystis carinii pneumonia in HIV-infected patients and atovaquone has been tested against murine toxoplasmosis in combination with pyrimethamine Pyrimethamine also has antibacterial value. Dapsone-pyrimethamine may prevent mycobacterial disease (M. tuberculosis, M. avium) in immunosuppressed patients infected with HIV.
Resistance to pyrimethamine, first reported shortly after its introduction in the 1950s, is now widespread worldwide. To delay (but not overcome) further malarial resistance to inhibitors such as pyrimethamine, combinations such as those above with sulfonamides or sulfones have been in use for some years. Fansidar, for instance, is currently the first-line drug of choice in a number of African countries, but resistance to both components of this combination is an increasing problem. The Douglas group have designed and synthesised molecules to overcome this resistance to pyrimethamine in a collaborative programme with Professor Worachart Sirawaraporn of Mahidol University in Thailand and Professor John Hyde of the University of Manchester.
Pyrimethamine inhibits dihydrofolate reductase-thymidylate synthase (DHFR-TS) in the folate biosynthetic pathway and resistance to it arises from mutation in the dhfr-ts gene. Studies on long‑term continuous culture isolates have shown that in the lethal species of the human malaria parasite, Plasmodium falciparum, resistance results in the first instance from an S108N mutation in the DHFR domain, but double and triple mutations impart much higher levels of resistance. One of the postgrads in the Douglas lab, Jim McKie, modelled the 3-dimensional structure for DHFR-TS from Leishmania major, although this is now superseded by an X-ray diffraction study of this enzyme. However, at the time of this study we did not have access to the coordinates. Even using the crude approximation of the L. major DHFR-TS model (as a basis for P. falciparum DHFR-TS), we were able to design inhibitors with the potential to overcome malarial pyrimethamine resistance. Other approaches the lab has taken against malaria include the inhibition of the parasite’s glutathione reductase enzyme (R. M. Lüönd, J. H. McKie, K. T. Douglas, M. J. Dascombe and J. Vale, J. Enz. Inhib.,(1998), 13, 327).
A rational drug design approach to overcoming drug resistance has been applied by the Douglas lab to pyrimethamine resistance in malaria pyrimethamine in a collaborative programme with Professor Worachart Sirawaraporn of Mahidol University in Thailand and Professor John Hyde of the University of Manchester.
A molecular model of the dihydrofolate reductase domain of DHFR-TS indicated for the S108N mutation that, if the active-site structure of Plasmodium DHFR-TS was similar to that of Leishmania, there would be a steric clash of the protein with the para-Cl atom of pyrimethamine POWER SL7. A suitable substituent in the adjacent meta position should avoid this clash and permit an additional interaction with the enzyme (J.H. McKie, K.T. Douglas, C. Chan, S.A.Roser, R. Yates, M. Read, J.E. Hyde, M.J. Dascombe, Y. Yuthavong and W.Sirawaraporn, J. Med. Chem. (1998) 41, 1367). Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR from Plasmodium falciparum, the Ki value of the meta-methoxy analog of pyrimethamine (CC83) was 1.07 nM but against the DHFR bearing the double mutation (C59R + S108N) the Ki values for pyrimethamine and meta-methoxy analogue were 71.7 and 14.0 nM, respectively. The meta-chloro analogue of pyrimethamine (S03) was a stronger inhibitor of both wild-type DHFR with (Ki 0.30 nM) and of the doubly-mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P.falciparum in vitro was also strongly inhibited by these compounds with fifty percent inhibition of growth occurring at 3.7 mM for the meta-methoxy and 0.6 mM for the meta-chloro compound with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 mM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P.berghei infected mice.
In the clinical situation, resistance is actually caused by a multiple mutation of the parasite DHFR (N51I + C59R + S108N + I164L) and in a second generation the teams have now produced analogues that overcome this multiple resistance (A. Sardarian et al., Org. Biomol. Chem (2003) 1, 960). The mBr compound showing Ki 5.1 nM against this mutant and IC50 37 mM against the resistant Plasmodium parasite growing in culture (compare > 5000 nM for pyrimethamine). Other groups of workers have followed this project and a crystal structure has recently been reported for the inhibitor S03 (that was discovered in our labs) bound to the plasmodial DHFR enzyme (Acta Cryst (2004) D60, 780-3).