Proc. Natl. Acad. Sci. USA Vol. 91, pp. 380-384, January 1994 Neurobiology Topography of a binding site for small amnestic peptides deduced from structure-activity studies: Relation to amnestic effect of amyloid f8 protein JAMES F. FLOOD*, EUGENE ROBERTStt, MARK A. SHERMAN§, BRUCE E. KAPLAN§, AND JOHN E. MORLEY* Geriatric Research Education and Clinical Center (GRECC), Veterans Administration Medical Center, St. Louis, MO 63106, and St. Louis University School of Medicine, Division of Geriatric Medicine, St. Louis, MO 63104; and tDepartment of Neurobiochemistry and §Biology Division, Beckman Research Institute of the City of Hope, Duarte, CA 91010 Contributed by Eugene Roberts, September 7, 1993 ABSTRACT Four peptides homologous to amyloid (B pro- With the exception of commercially purchased peptides tein containng the Val-Phe-Phe (VFF) sequence administered VF and FV, all peptides used in these studies were synthe- intracerebroventricularly after training caused amnesia for sized and analyzed to establish purity by standard methods. footshock active avoidance training in mice. Results with VFF Prior to neutralization and appropriate dilution with saline, and other peptides containing VFF or portions thereof were peptides were dissolved in (i) saline, (ii) dimethyl sulfoxide, used to generate a topographic map for a hypothetical binding (iii) glacial acetic acid, or (iv) NaOH. Upon testing for surface for amnestic peptides, termed Z. Effects on retention of retention of FAAT, groups receiving posttraining icv admin- footshock active avoidance training were rationalized in terms istration of2 1d ofappropriate dilutions ofthe above vehicles of flt to Z, making possible design of potential memory- showed no significant differences among them (ANOVA, F modulating peptidic and nonpeptidic substances. Three pep- < 1). The mean trials to criterion of the most frequently tides that neither improved nor impahred retention blocked the emploved vehicle (vehicle ii above; final concentration of amnestic effects of -(12-28), a peptide homologous to amyloid 13 protein, opening the way to development of substances that dimethyl sulfoxide, 0.001%) were compared to that of mice can antagonize the neurotoxic effects of amyloid (3 protein on receiving test peptides. neural structures and thus attenuate symptoms and progres- With the exception of dose-response curves, the experi- sion of Alzheimer disease. ments reported below tested whether or not peptides im- paired retention at 6.14 nmol per mouse. Although f-(1-28) Intracerebroventricular (icv) administration of a synthetic (1) and (-(12-28) were significantly amnestic at 1.5 nmol per peptide homologous to amyloid 13protein (AP), [Glnl1]13-(1- mouse, the 6.14-nmol dose was chosen so that substances 28) (DAEFRHDSGYQVHHQKLVFFAEDVGSNK), im- with weaker effects might be detected. mediately after training caused amnesia for footshock active avoidance training (FAAT) in mice in a dose-dependent RESULTS fashion. Also amnestic were peptides l3-(12-28), -(18-28), and /3-(12-20) (1). The above amnestic peptides have the Topography of a Binding Site Deduced from a Structure- sequence VFF in common. ,B-(1-11), which does not contain Activity Study of Amnestic Effects. Examination of Corey- VFF, is not amnestic (see below). FAAT experiments were Pauling-Koltun (CPK) models of the peptides tested (Table performed with 28 peptides, 22 of which contained the VFF 1) and superimposition of appropriate configurations of the sequence or portions thereof. A topographic model for the amnestically active ones led to devisal of a provisional binding site ofamnestic peptides, termed Z, was derived from topographic model ofthe site (Z) to which all ofthe amnestic the structure-activity study of the latter. substances might bind. A more refined hypothetical three- In separate experiments, 3 of 15 nonamnestic peptides dimensional binding surface for the peptides (described be- were found to counteract the amnestic effects of ,B-(12-28). low) then was generated by surrounding with small molecules that mimic the properties of amino acids the peptide Gln- MATERIALS AND METHODS Phe-Phe-y-aminobutyric acid that fit optimally to all of the proposed contact points on the original map. A solvent- The care and handling of test mice, surgical procedures for accessible surface was generated using the algorithm of preparation of mice for icv administration, the testing appa- Connolly (2). Peptides then were docked onto the surface and ratus, training and testing procedures, and methods of sta- energy-minimized to optimize contacts [Dreiding II forcefield tistical treatment of data have been described (1). Immedi- (3)]. Images were created with the BIOGRAF program (Mo- ately after training, groups of mice (10-15 mice per group) lecular Simulations, Waltham, MA). The hypothetical recep- were given icv injections (2 ,ul) of vehicle alone or vehicle tor map thus generated (Z) is presented at the outset. Without containing 6.14 nmol of a peptide. Tests for retention of FAAT were performed 1 week later. Results were expressed a detailed molecular definition of the structure of Z, quanti- as the mean and SEM. Significance of overall effects of tative assessments of binding energies of ligands are not treatment was determined by ANOVA run on trials to reach possible. Qualitative and semiquantitative arguments devel- a footshock avoidance criterion in the retention test (trials to oped with the data at hand were employed to interpret the criterion). Tukey's t test was used to make statistical com- results. Numbering clockwise from the lower left in Fig. 11, parisons among group means. Treatment was judged to be the postulated sites on Z are characterized as: 1, H-bonding; amnestic when the mean of vehicle and a treatment group gave a P value of <0.01. Abbreviations: AA3, amyloid ,B protein; FAAT, footshock active avoidance training; icv, intracerebroventricular; VIP, vasoactive intestinal peptide. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Neurobiochemistry, Beckman Research Institute of the City of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Hope, Duarte, CA 91010. 380 Downloaded by guest on September 23, 2021 Neurobiology: Flood et al. Proc. Natl. Acad. Sci. USA 91 (1994) 381 Table 1. Effect of 6.14 nmol of icv-administered peptides on amino group, for a total of five groups per molecule. Water retention of T-maze FAAT (15 mice per group) clusters may be organized at those sites, the sizes and shapes Peptide Trials to of which are determined by combinatorial influences of Peptide tested criterion, no. molecular and environmental factors. Unbound Z site is presumed to have water clusters associated with sites 1, 3, 1* VFF 9.67 0.40t and 5 (Fig. 11). VFF (Fig. 14) is posited to orient on Z in such 2* VFFAE 8.73 0.38t a manner that the attractive energy between VFF and Z 3* KLVFF 7.73 0.42 becomes great enough to squeeze out intervening water, in 4* KLVFFAE 6.87 0.27 this way minimizing the energy of the system. The molar 5* FFV 9.07 0.41t amnestic efficacy ofVFF was not significantly different from 6t FFVG 8.47 0.33t+ that ofthe APhomologue [Gln11]f-(1-28) (Fig. 2A). Ifbinding 7t DFFV 7.27 0.38 of its VFF segment to Z is key to the amnestic effect of the 8t DFFVG 6.87 0.33 latter, its flanking regions must contribute to strength of 9 KLFFVAE 9.58 0.39t attachment since neither the a-amino group of Val nor the lo§ VF 9.13 0.94t carboxyl group of F-2 is available to contribute to binding ii§ VFT 9.73 0.31t efficacy. Similar considerations apply to other such compar- 12§ AVFT 9.73 ± 0.21t isons, as for AVFT and VIP in Fig. 2B. 13§ AVF 6.80 ± 0.25 If residues with H-bonding side chains that do not bind to 14 VVF 9.27 ± 0.37t Z are added at either end of a peptide that, by itself, binds to 15 VFV 9.07 ± 0.43t Z, water clusters on the nonbinding moieties may become 16 FVF 6.93 ± 0.29 sufficiently large and stable so that resulting physical and 171 FV 9.40 ± 0.46t energetic barriers deter effective attachment ofthe peptide to 1811 SFVG 7.47 ± 0.45 Z. A curvilinearly decremental trend in amnestic potency was 19** QFVG 6.80 ± 0.39 noted with increasing numbers of side-chain H-bonding 20ft FF 7.00 ± 0.42 groups in such peptides related to VFF (Fig. 3A). 21#* QFFG 9.00 ± 0.46t FFV, also amnestic (Table 1 and Fig. 3B), attaches to 22 SFFG 7.33 ± 0.40 aromatic sites 2 and 4 on Z, but the C-terminal carboxyl group 23§H AFIG 6.67 ± 0.26 of Val in FFV is directed to cationic site 3 (Fig. 17). Addition 24§§ AIFT 7.00 ± 0.29 of H-bonding groups to FFV also reduced amnestic potency 25§§ GFMT 6.80 ± 0.34 (Fig. 3B). It is presumed that a water cluster can be mobilized 26§§ NLIT 7.07 ± 0.36 on the amide nitrogen of a peptide bond when Gly is the 27 vv 6.80 ± 0.31 C-terminal residue because, uniquely among the amino acids, 28* DAEFRHDSGYQ 6.93 ± 0.28 the C-terminal Gly offers little side-chain interference to Data for trials to criterion are expressed as the mean t SEM. P water cluster formation. For the latter reason, the number of values were obtained for selected comparisons after obtaining a H-bonding groups assigned to a peptide was increased by one significant F value by ANOVA; F(16,204) = 8.22, P < 0.001. for peptides with a C-terminal Gly (Fig.