DENDRIMER CATECHOL

~

^{~ O H}

and fidelity.

polymer matrix. These interior regions possess a surface area approximately equivalent to the amount found on the exterior of the polymer spheroid. Simi- larly, the volume associated with these solvent-filled void spaces is ~50% of that contained within the full spheroid. These internal regions can be used for seques- tering small molecules, as illustrated by the example of the dopamine-loaded and catechol-modified delivery dendrimer. Materials of this type could be designed to encapsulate selected target molecules or to contain sites for modification to produce catalytically active buried pockets.

The penta-erythritol polyether dendrimers were found to possess structures and properties quite different from those of the polyamidoamine series. The structures adopted by the polyethers are dense and spherelike early in the poly- mer growth cascade. Only negligible amounts of interior surface area and volume were found for these materials. Estimates of the amount of surface area per hy- droxyl head group indicated that this generation 4 dendrimer should not be structurally stable, consistent with experimental observation.

(1.) D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, P. Smith. Polym. J., Tokyo, 1985, 17, 117.

(2.) D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P. Smith. Macromolecule., 1986, 19, 2466.

(3.) D.A. Tomalia, M. Hall, D.M. Hedstrand. J. Am. Chem. Soc. 1987, 109, 1601.

(4.) D. A. Tomalia, V. Berry, M. Hall, D. M. Hedstrand. Macromolecule.,, 1987,

!O, 1164.

(5.) H. Hall, A. Padias, R. McConnell, D. A. Tomalia. J. Org. Chem., 1987, 5!, 5305.

(6.) P.B. Smith, S. J. Martin, M. J. Hall, D. A. Tomalia. Applied Polymer AnalyJia and Characterization. Edited by J. Mitchell, Jr. Hanser Publishers:

New York (1987), p. 357.

(7.) D. A. Tomalia, D. M. Hedstrand, L. R. Wilson. Kirk-Othmer Encyclopedia of Polymer Science and Engineering, 1989, in press.

(8.) L. Wilson and D. Downing, personal communication.

(9.) POLYGRAF is an interactive molecular mechanics/graphics program avail- able from Molecular Simulation, Inc. Pasadena, CA. All simulations were performed on DEC VAX 8650 and Alliant FX8/8 computers, and structures were displayed on Evans & Sutherland PS330 and PS390 series graphics systems.

(10.) S. J. Weiner, P.A. Kollman, D. A. Case, U. C. Singh, C. Ghio, G. Alagona, S. Profeta, Jr. P. Weiner. J. Am. Chem. Soc. 1984, 106, 765.

(11.) F. M. Richards. Ann. Rev. Biophya. Bioeng. 1977, 6, 151.

(12.) M. L. Connolly. J. Appl. Cry.,t. 1983, 16, 548.

calculated values was less than 0.1 % but the cpu utilization had increased by a factor of 3 (NDIV=4) or 10 (NDIV=5). Hence, it was determined that the recommended value of NDIV =3 was most suitable.

(15.) A.M. Naylor, W.A. Goddard III, G. E. Keifer, D.A. Tomalia. J. Am. Chem.

Soc., 1989, in press.

(16.) PLOTIT Interactive Graphics and Statistics available from Scientific Pro- gramming Enterprises, Haslett MI.

(17.) C. Tan.ford, The Hydrophobic Effect. A Formation of Micellea and Biological Membranea. Wiley Interscience: New York (1980).

(18.) J. W. Kebabian, T. Agui, J. C. van Oene, K. Shigematsu, J. M. Saavedra.

Trenda in Pharmacological Science., 1986, 96.

Section III

Dihydrofolate Reductase:

Molecular Simulation of Wild-type and

Mutant Enzyme Complexes

Portions of the text of this section will compose an article coauthored with William A. Goddard III and Stephen J .Benkovic. It is to be submitted to the

Journal of the American Chemical Society.

Molecular modeling and simulation tools were used to investigate various Dihydrofolate Reductase (DHFR) complexes.

Three distinct sources of DHFR were analyzed in homology studies. The sequence homology between E. coli and L. ca.,ei proteins is less than 30%. Our investigations indicate that the chemical homology for the catalytic pocket is

~70%, explaining the remarkable similarity in their steady-state kinetic profiles.

The inclusion of Chicken DHFR yields only ~60% chemical homology with the bacterial forms, suggesting subtle differences in the character of the active site.

This molecular modeling and subsequent simulation were used to engineer E.

coli• Chicken hybrid enzymes targeted to reduce folate.

The structure and properties of DHFR complexes (E. coli, single-site mu- tants, and Chicken loop E. coli hybrids) were studied using molecular dynamics simulations. The calculations on the wild-type E. coli enzyme indicate: (a) the energetically favored conformation of bound methotrexate's (MTX) pteridine ring is that found in the crystal studies; (b) the active site region accommodates either the xtl or the anti form of both MTX and dihydrofolate (DHF); and ( c) the Anti-DHF ternary complex is favored for catalysis with a ground-state hydride transfer distance of ~3.6

A.

The investigations on single-site mutants revealed structural changes that explain the experimental kinetic data. Phe-31-+Tyr-31 introduces reorientation of substrate binding that is consistent with the observed increase in the product off-rate (the rate-limiting step). Modifications of the alkyl side chain at position 54 (Leu-54-+Ile-54 or Leu-54-+Gly-54) increase the separation between the co- factor (nicotinamide) and substrate (pteridine) reaction centers, thus increasing the rate of hydride transfer sufficiently so that it becomes rate-limiting.

an ideal system for our interests, which are to examine how changes in the active site of an enzyme affect function and reactivity.

Dihydrofolate Reductase (DHFR) catalyzes the reduction of 7,8- dihydrofolate (DHF; 1) to 5,6,7,8-tetrahydrofolate (THF; 2), as depicted below.

Pteridyl moiety

1

DHFR

2

This reduction requires: (1) a hydride (H-) to be transferred to the substrate from a nicotinamide adenine dinucleotide phosphate (N ADPH) cofactor source that is bound in a ternary complex with DHFR and DHF, and (2) a proton (H+) to be supplied from some source (possibly H20). Figure 1 illustrates the DHFR,DHF,NADPH ternary complex. This structure is one of the products of the work described in this document.

The product THF is an important coenzyme in a number of one-carbon transfer reactions. Its most important role, though, is in the synthesis of thymidy- late (dTMP) from deoxyuridylate (dUMP) by thymidylate synthetase. In this metabolic process, THF not only is the source of a carbon fragment but is also used as a reductant. Hence, it is used in substrate, rather than coenzyme, quan-

Figure 1. The active site of wild-type E. coli Dihydrofolate Reductase with dihydrofolate a.nd NADPH (both shown in red) bound. The solvent surface of the active site is mapped out by the series of dots.

Figure 3. Wild-type E. coli DHFR with MTX (red) and NADPH (light blue) bound in their respective sites. Asp-27 (dark blue), involved in protonation of the substrate pteridine ring, is shown salt-bridged to the MTX. Phe-31 (green) a.nd Leu-54 (blue) are strictly conserved residues that form part of the substrate binding site.

Figure 1.

Figure 3.

metabolic cycle is depicted in Figure 2. Inhibition of DHFR leads to a deficiency of dTMP and ultimately to a disruption of nucleic acid biosynthesis. It is this mechanism that is the biochemical basis for the antifolate drugs that have found therapeutic applications.¹

The known inhibitors of DHFR may be divided into classical and nonclassical subsets. The classical inhibitors are closely related structurally to the folic acid natural substrates of DHFR. Thus, they are expected to bind to the enzyme in a manner quite similar to normal substrates but cannot be easily reduced, preventing the DHFR from transforming DHF to THF. Methotrexate (MTX; 3), shown below, is one important example of a classical DHFR inhibitor.

H

I

H2N~*N N l O CO2

, ,N ''

I A

^____F\__

II I

: ~ , N CH2-N~C-N-C-CH2-CH2-CCJi

'-

_NH

-.

_{I ,}

-1-

_'

I I

', 2 , , CH3 , H H

, I \ I

~ -^{_. ' ~}

3

Many nonclassical inhibitors, chemically distinct from the folic acid derivatives, have been identified.^{1 •2} These agents may well bind to the enzyme in a manner quite different from that of the natural substrates.

Recent advances in experimental methodologies have made possible the in- vestigation of structure-function relationships in this enzyme system. The crystal structure of DHFR from two bacterial sources (E. coli and L. ca.,ei) has been solved, refined to a 1. 7

A

resolution, ³-e and made available tp the scientific com- munity through the Brookhaven Protein Database. The study of DHFR from L. ca.,ei involved the ternary complex of enzyme, MTX (inhibitor), and NADPH (cofactor). The crystal structure of E. coli DHFR was determined as a dimer of

Figure 2. Metabolic cycle encompassing the reduction of DHF to THF by DHFR.

THF