Picomolar Transition State Analogue Inhibitors of Human 5′-Methylthioadenosine Phosphorylase and X-ray Structure with MT-Immucillin-A

Vipender Singh, Wuxian Shi, Gary B. Evans, Peter C. Tyler, Richard H. Furneaux, Steven C. Almo, Vern L. Schramm

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65 Scopus citations


Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase ( and spermine synthase ( Inhibition of polyamine synthesis is a validated anticancer target. We designed and synthesized chemically stable analogues for the proposed transition state of human MTAP on the basis of the known ribooxacarbenium character at all reported N-ribosyltransferase transition states [Schramm, V. L. (2003) Acc. Chem. Res. 36, 588-596]. Methylthio-immucillin-A (MT-ImmA) is an iminoribitol tight-binding transition state analogue inhibitor with an equilibrium dissociation constant of 1.0 nM. The immucillins resemble the ribooxacarbenium ion transition states of N-ribosyltransferases and are tightly bound as the N4′ cations. An ion pair formed between the iminoribitol cation and phosphate anion mimics the ribooxacarbenium cation-phosphate anion pair formed at the transition state and is confirmed in the crystal structure. The X-ray crystal structure of human MTAP with bound MT-Imm-A also reveals that the 5′-methylthio group lies in a flexible hydrophobic pocket. Substitution of the 5′-methylthio group with a 5′-phenylthio group gives an equilibrium binding constant of 1.0 nM. Methylthio-DADMe-immucillin-A is a pyrrolidine analogue of the transition state with a methylene bridge between the 9-deazaadenine group and the pyrrolidine ribooxacarbenium mimic. It is a slow-onset inhibitor with a dissociation constant of 86 pM. Improved binding energy with DADMe-immucillin-A suggests that the transition state is more closely matched by increasing the distance between leaving group and ribooxacarbenium mimics, consistent with a more dissociative transition state. Increasing the hydrophobic volume near the 5′-position at the catalytic site with 5′-phenylthio-DADMe-immucillin-A gave a dissociation constant of 172 pM, slightly weaker than the 5′-methylthio group. p-Cl-phenylthio-DADMe-immucillin-A binds with a dissociation constant of 10 pM (Km/Ki* value of 500000), the tightest binding inhibitor reported for MTAP. These slow-onset, tight-binding transition state analogue inhibitors are the most powerful reported for MTAP and have sufficient affinity to be useful in inhibiting the polyamine pathway.

Original languageEnglish (US)
Pages (from-to)9-18
Number of pages10
Issue number1
StatePublished - Jan 13 2004

ASJC Scopus subject areas

  • Biochemistry


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