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

Research output: Contribution to journalArticle

58 Scopus citations

Abstract

Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase (2.5.1.16) and spermine synthase (2.5.1.22). 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
JournalBiochemistry
Volume43
Issue number1
StatePublished - Jan 13 2004

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this

Picomolar Transition State Analogue Inhibitors of Human 5′-Methylthioadenosine Phosphorylase and X-ray Structure with MT-Immucillin-A. / Singh, Vipender; Shi, Wuxian; Evans, Gary B.; Tyler, Peter C.; Furneaux, Richard H.; Almo, Steven C.; Schramm, Vern L.

In: Biochemistry, Vol. 43, No. 1, 13.01.2004, p. 9-18.

Research output: Contribution to journalArticle