Development of a Pharmacophore Model for a Novel Hematoregulatory Peptide Pradip K. Bhatnagar,* ,† Doreen Alberts, † James F. Callahan, † Dirk Heerding, † William F. Huffman, † Andrew G. King, ‡ Steve LoCastro, † Louis M. Pelus, ‡ and Joanne S. Takata † Departments of Medicinal Chemistry and Molecular Virology and Host Defense SmithKline Beecham Pharmaceuticals King of Prussia, PennsylVania 19406 ReceiVed August 19, 1996 Hematopoietic growth factors play a pivotal role in orches- trating cellular host defense mechanisms against bacterial, fungal, and viral infections. The utility of these agents lies in their ability to stimulate nonspecific host defense by increasing immune cell numbers and enhancing the host effector cell function. 1 Although protein therapeutics have proven beneficial, their chronic administration still poses challenges such as immunogenicity and oral delivery. Therefore, structurally simple, low molecular weight compounds that either mimic or induce endogenous growth factors are attractive targets for drug design. We have reported on the peptide 1 (SK&F 107647, Figure 1) that stimulates proliferation of murine and human granulocyte-macrophage colony-forming units (CFU-GM) in vivo, enhances bone marrow stem cell engraftment during bone marrow transplant, and increases antimicrobial activity of macrophages and polymorphonuclear (PMN) cells. 2 In infection models, SK&F 107647 (1) enhances the survival of mice infected with either Candida albicans or Herpes simplex type 2 virus. 3,4 Prophylactic administration of 1 (both alone and in combination with antibiotics) to rats infected with a fibrin- thrombin clot containing either Gram-negative (Escherichia coli) or Gram-positive (Streptomyces aureus) bacteria enhances survival. 5 SK&F 107647 (1) manifests its biological response by inducing hematoregulatory growth factors from bone marrow stromal cells. 6 The paucity of information about the putative receptor for 1 has limited our ability to rationally design non-peptidic or peptidomimetic analogs for this compound. A detailed structure- activity relationship (SAR) study of 1 and its analogs has shown quite specific requirements for biological activity in the colony- stimulating activity (CSA) assay. 2 The C-terminal carboxylates of 1 can be replaced with carboxamides without loss of activity, and Glu3 can be replaced with Asp or Ser suggesting that a charged side chain is not required at this position. There is a stringent requirement for an acidic residue at position 3 where Asp was replaceable only with Glu. This suggests that this residue probably forms a critical ionic interaction with a complementry basic residue. The amino group of lysine at position 5 as well as its spatial location is critical for the biological activity, since analogs containing lower homologs of lysine, such as ornithine and 2,4-diaminobutyric acid, or any other amino acid were virtually inactive at the highest dose tested (1 mg/mL). This again suggests that the Lys5 residue is involved in a critical ionic interaction. The SAR for the spacer (position 4), connecting the two halves of the molecule, is even more remarkable. The number of methylene units spanning the diaminodicarboxylic acids at position 4 is critical. The di- and tetramethylene spacers (diaminosuberic acid and diaminoadipic acid) are well tolerated; whereas, the mono-, tri-, penta-, and hexamethylene spacers (diaminoglutaric acid, diaminopimelic acid, diaminoazelaic acid, and diaminosebacic acid) are not. If one assumes that the bridge adopts an extended conformation, an even-membered alkylene spacer could place the two peptide chains in a “syn” relationship, while an odd-membered spacer would orient them in an “anti” relationship (Figure 2). It is possible that this type of confor- mational bias plays a role in determining the biological activity of a given analog. The vast difference in the EC 50 of analogs containing even- and odd-membered spacers suggests that the distance and the relative orientation the two peptide chains are crucial for biological activity. Considering this data, we propose a model where the side chains of the Asp3 and Lys5 residues are involved in intramo- lecular ionic interactions creating salt bridges and, along with the methylene spacer of residue 4, present residues 1 and 2 to the putative receptor. Two possible permutation of the model which include intramolecular salt bridges are shown in Figure 3. To test this model, the peptide analog 2 was synthesized where both the Asp3and Lys5 were replaced with Glu3and Orn5. These modifications change the length of the side chains of both residues 3 and 5 without affecting the overall size of the pseudoring formed by the intramolecular salt bridges. Analog * Author to whom correspondence should be addressed: Pradip K Bhatnagar, Departments of Medicinal Chemistry, Mail Code UM 2412, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King Of Prussia, Pennsylvania 19406; telephone (610) 270-6616; fax (610) 270-4162. † Department of Medicinal Chemistry. ‡ Department of Virology and Host Defense. (1) Sachs, L. Proc. Natl. Acad. Sci. U.S.A. 1996, 19, 4742-4749 and other references cited in this review. (2) Bhatnagar, P. K.; Agner, E.; Alberts, D.; Arbo, B. A.; Callahan, J.; Cuthbertson, A.; Engelsen, S. J.; Hartmann, M.; Heerding, D.; Hiebl, J.; Huffman, W. F.; Hysben, M.; Fjerdingstad, H.; King, A. K.; Kwon, C.; Kremminger, P.; LoCastro, S.; Løvhaug, D.; Pelus, L. M.; Petteway, S.; Takata, J. J. Med. Chem. 1996, 39, 3814-3819. (3) DeMarsh, P. L.; Sucoloski, S. K.; Frey, C. L.; Koltin, Y.; Actor, P.; Bhatnagar, P. K.; Petteway, S. R. Immunopharmacol 1994, 27, 199-206. (4) Pelus, L. M.; DeMarsh, P. L.; King, A. G.; Frey, C.; Bhatnagar, P. K. Colloq. INSERM 1993, 229, 193-200. (5) DeMarsh, P. L.; Wells, G. I.; Lewandowski, T. F.; Bhatnagar, P. K.; Ostovic E. J. J. Infect. Dis. 1996, 173, 205-211. (6) Pelus, L. M.; King, A. G.; Broxmeyer, H. E.; DeMarsh, P. L.; Petteway, S. R.; Bhatnagar, P. K. Exp. Hematol. 1994, 22, 239-247. Figure 1. Structure and numbering system for SK&F 107647 (1). Figure 2. Schematic of a,a′ substituents on the odd- and even- membered “methylene bridge” containing residue at position 4. 12862 J. Am. Chem. Soc. 1996, 118, 12862-12863 S0002-7863(96)02919-8 CCC: $12.00 © 1996 American Chemical Society