C O M M U N I C A T I O N S
Table 1. Dissociation Constants for Transition State Analogue
Inhibitors of Purine Nucleoside Phosphorylase
3 suggest that its residence time on PNP in erythrocytes is in excess
of tissue lifetime, thus capture of additional transition state binding
13
16
energy is unnecessary for inhibitor design purposes.
The simplified inhibitor compound 8, though having higher K
values than the other compounds in Table 1, has less than 350-
fold higher K value than 3. DADMe-Immucillin-H [3] is currently
d
d
being evaluated for T-cell immunosuppression and is in phase I
clinical trials (http://www.biocryst.com/pipeline.htm). Compound
8
is especially novel in that it has no stereogenic centers, but its
geometric similarity to the HsPNP transition state permits prefer-
ential binding to HsPNP.
The geometric differences between the Immucillins and the
DADMe-Immucillins is sufficient to distinguish between the
transition states of the bovine and human enzymes, even with the
removal of all hydroxyl and hydroxymethyl groups from the
hydroxyl pyrrolidine. These compounds can distinguish between
two enzymes which have 87% sequence identity and have totally
conserved active site residues, both in identity and position in the
catalytic site.1 The ability to distinguish between enzymes with
such great homology highlights the power of transition state
determination and the subsequent synthesis and use of transition
state analogues.
7,18
Acknowledgment. This work was supported by the NIH
Research Grant Nos. GM41916 and AI49512.
Supporting Information Available: Synthetic methods, spectro-
scopic and analytical data for compounds 7 and 8. Assay conditions
for K determination for Hs- and BtPNPs. This material is available
d
free of charge via the Internet at http://pubs.acs.org.
References
(
1) Stoeckler, J. D. Purine Nucleoside Phosphorylase: A Target for Chemo-
therapy. In DeVelopments in Cancer Chemotherapy; Glazer, R. J., Ed.;
CRC Press: Boca Raton, FL, 1984; pp 35-60.
(
2) Bantia, S.; Kilpatrick, J. M. Curr. Opin. Drug DiscoVery DeV. 2004, 7,
243-247.
(
3) (a) Schramm, V. L. Methods Enzymol. 1999, 308, 301-355. (b) Schramm,
V. L. Acc. Chem. Res. 2003, 36, 588-596. (c) Wolfenden, R. Annu. ReV.
Biophys. Bioeng. 1976, 5, 271-306.
(
4) Rodgers, J.; Femec, D. A.; Schowen, R. L. J. Am. Chem. Soc. 1982, 104,
3263-3268.
(
(
(
(
(
5) Cleland, W. W. Methods Enzymol. 1982, 87, 625-641.
6) Berti, P. J. Methods Enzymol. 1999, 308, 355-397.
7) Kline, P. C.; Schramm, V. L. Biochemistry 1993, 32, 13212-13219.
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9) Furneaux, R. H.; Tyler, P. C. J. Org. Chem. 1999, 64, 8411-8412.
constants, clearly demonstrate that the differences in inhibitor
geometry between these two compounds is sufficient to result in
binding affinity changes, where the Immucillin analogue [7] has a
greater binding affinity for BtPNP and the DADMe-Immucillin
analogue [8] binds more tightly to HsPNP. The differential binding
of these inhibitors on the basis of their different transition state
structures allows compounds 1-8 to be used as tools to derive
insight into the relative transition state position of uncharacterized
ribosyl transferases.
(10) Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm,
V. L. Biochemistry 1998, 37, 8615-8621.
(
11) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.; Tyler,
P. C. J. Med. Chem. 2003, 46, 5271-5276.
(12) The arsenate, which is only found in van der Waals contact with the ribose
in both transition states, was not displayed for simplicity of visualizing
the ribose base geometry changes between the two enzymes.
13) Inhibition constants are reported from the indicated literature value with
the least error. If no reference is given for a value, it is previously
unreported. Dissociation constants represent values corresponding to slow-
onset, tight-binding inhibition, except for compound 7 for HsPNP and
compounds 7 and 8 for BtPNP. Values shown here are final equilibrium
dissociation constants.
(
Although 1-4 are powerful inhibitors, it is not possible to
generate chemically stable analogues that perfectly mimic unstable
transition states. Human and bovine PNPs provide a rate enhance-
(
14) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.; Tyler,
12
ment of approximately 10 -fold over the uncatalyzed reaction.
P. C. J. Med. Chem. 2003, 46, 3412-3423.
-
5
Therefore, since the K
d
for inosine is approximately 10 M, the
(15) Lewandowicz, A.; Taylor Ringia, E. A.; Ting, L.-M.; Kim, K.; Tyler, P.
C.; Evans, G. B.; Zubkova, O. V.; Mee, S.; Painter, G. P.; Lenz, D. H.;
Furneaux, R. H.; Schramm, V. L. J. Biol. Chem. 2005, 280, 30320-30328.
predicted binding affinity for a transition state analogue with perfect
-
17
3
mimicry would be 10
with HsPNP to give a K
×
M. The best inhibitor of Table 1 is 4
(
16) Lewandowicz, A.; Tyler, P. C.; Evans, G. B.; Schramm, V. L. J. Biol.
-
12
d
of 7 × 10 M, which corresponds to 7
Chem. 2003, 278, 31465-31468.
5
(17) Fedorov, A.; Shi, W.; Kicska, G.; Fedorov, E.; Tyler, P. C.; Furneaux, R.
H.; Hanson, J. C.; Gainsford, G. J.; Larese, J. Z.; Schramm, V. L.; Almo,
S. C. Biochemistry 2001, 40, 853-860.
10 -fold weaker binding than that of a perfect transition state
6
mimic. Compound 4 binds 5 × 10 -fold tighter than substrate,
thereby utilizing over half of the potential binding energy afforded
by the enzymatic rate acceleration (kcat/knon) and capture of transition
state features. Physiological experiments in mice with compound
(18) Shi, W.; Lewandowicz, A.; Tyler, P. C.; Furneaux, R. H.; Almo, S. C.;
Schramm, V. L. Unpublished data (pdb file name 1RT9).
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J. AM. CHEM. SOC.
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