Synthesis of 5‘-Methylthio Coformycins:  Specific Inhibitors for Malarial Adenosine Deaminase

Abstract

Transition state theory suggests that enzymatic rate acceleration (k cat /k non ) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Since transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2′-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5'-methylthioadenosine. In contrast, human adenosine deaminase (HsADA) does not deaminate 5'-methylthioadenosine. 5′-Methylthio coformycin and 5'-meththio-2′-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to cause toxicity in humans and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.

Structures of Substrate- and Inhibitor-Bound Adenosine Deaminase from a Human Malaria Parasite Show a Dramatic Conformational Change and Shed Light on Drug Selectivity

“…Comparison of the now available ligand-bound form of plasmodial ADA, represented by the P. vivax structures, to the ligand-bound mammalian structures, however, indicates that the structural differences suggested by Tyler et al 7 do not actually have a significant effect on the pocket size in the bound state. The indicated mammalian Cys to plasmodial Ile substitution is located at the beginning of the β3/α12 Fig.…”

“…The structure of the closed Plasmodium ADA (PDB ID 2PGF) superimposes 10 on the closed MmADA (PDB ID 1ADD) with an RMSD of 1.74 Å for 308 aligned C α atoms and on the closed BtADA (PDB ID 1KRM) with an RMSD of 1.75 Å for 308 aligned C α atoms. It has not been clear from the available structures how the alternate substrate, MTA, 4 or the specific substituted inhibitors, 5′-methylthio-DCF (5′-MeS-DCF), 5′-propylthio-DCF (5′-PrS-DCF), and 5′-phenylthio-DCF (5′-PhS-DCF) 7 bind selectively to the plasmodial enzyme over the mammalian counterparts.…”

“…7 In particular, the structural changes implicated are a 1 Å shift in the backbone away from the substrate at conserved Tyr128 and an additional backbone shift in the vicinity of Ile170 and Asp172 of plasmodial ADA 7 (residue numbering is from PvADA). The shift at this second location was attributed to substitutions of Cys153 to Ile170 and Met155 to Asp172 between mammalian and plasmodial ADA sequences (Fig.…”

“…Indeed, knowledge of this expanded activity has led to the recent design of a series of 5′-substituted deoxycoformycins that selectively inhibit the P. falciparum enzyme but not the human or bovine enzymes. 7 Insight into the structural basis of this selectivity has been lacking, however.…”

Purine Salvage Pathways in the Intraerythrocytic Malaria Parasite Plasmodium falciparum

“…The 5Ј-methylthio coformycins inhibit PfADA at very low concentrations but do not inhibit hADA (63). The abilities of these compounds to inhibit parasite growth in the presence of physiologically relevant concentrations of inosine and hypoxanthine remain to be established.…”