Development of a pharmacophore model for a novel hematoregulatory peptide

Full text of DOI:10.1021/ja962919h

12862
J. Am. Chem. Soc. 1996, 118, 12862-12863
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
Figure 1. Structure and numbering system for SK&F 107647 (1).
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
Figure 2. Schematic of a,a′ substituents on the odd- and even-
membered “methylene bridge” containing residue at position 4.
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 EC50 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.
S0002-7863(96)02919-8 CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12863
Figure 4. Colony-stimulating activity of compound 1 [b] and
compound 4 [O] on C6.4 cells.
Table 1. EC50 of 1 and Its Analogs in the CSA Assay
compound
structurea
EC50 (pM)
1
2
3
4
(pGlu-Glu-Asp)2-Sub-(Lys-OH)2
(pGlu-Glu-Glu)2-Sub-(Orn-OH)2
(pGlu-Glu-Asp)2-Sub-(Orn-OH)2
(pGlu-Glu-â-Ala)2-Dab
4
4
nab
684
a Sub ) (2-S,7-S)-2,7-diaminosuberic acid; Dab** ) 1,4-diami-
nobenzene. b na not active at 1 mg/mL.
Figure 3. Hypothetical models for the side chain interactions of 1.
Scheme 1. Scheme for the Synthesis of Compound 2a
The novel peptidomimetic 4, designed on the basis of this
hypothesis, represents a major improvement over the structure
of 1. Evaluation of 4 in the CSA assay indicated that this
compound retains 100% of activity of 1 (Figure 4). Although
the potency of 4 does not match the potency of 1, it is still a
very potent hematoregulatory molecule with an EC50 in sub-
nanomolar range (Table 1). This data is especially impressive
since over half of the molecular mass and six asymmetric centers
have been eliminated. These results further validate the
pharmacophore model and allow for the refinement of our
knowledge of the recognition event. Specifically, the results
support that residues 1 and 2 are important for the recognition
event and that residues 3-5 serve to orient residues 1 and 2
into their biologically active conformation. These data also
imply that the achiral spacer of 4 (a) may not be the optimal
replacement for residues 3-5 and (b) is conformationally too
flexible to allow differentiation of the “antiparallel” or “parallel”
pharmacophore models.
In conclusion, we have proposed a pharmacophore model for
the interaction of SK&F 107647 (1) and its analogs with their
putative “receptor”. This model is supported by the data on
several key peptide analogs. In addition, we have designed and
synthesized a biologically active peptidomimetic analog validat-
ing many aspects of the proposed model. These results show
that it is possible to use this model to rationally design
structurally simple, low molecular weight molecules that
manifest many of the biological effects seen by hematopoietic
growth factors.
a (a) Boc-â-Ala, EDC‚HCl, HOBt, DIEA, DMF. (b) TFA, CH2Cl2.
(c) Boc-Glu(OBz), BOP, HOBt, DIEA, DMF. (d) TFA, CH2Cl2. (e)
pGlu, BOP, HOBt, DIEA, DMF. (f) HF, 0 °C.
2 was shown to be equipotent to 1, while analog 3, which
contains a smaller pseudoring structure, was inactive. These
results are consistent with the proposed model where the side
chains of residues 3 and 5 interact ionically to form salt bridges
(either intrapeptide chain or interpeptide chain) with each other,
locking a biologically active conformation (Figure 3).
We reasoned that if residues 3-5 serve to lock a specific
biologically active conformation and present residues 1 and 2
in the correct orientation, then it should be possible to replace
this entire scaffold with a simpler structure. To test this
hypothesis a series of compounds was synthesized in which
residues 3-5 were excised and replaced with various spacers.
While most of these changes resulted in inactive molecules,
incorporation of N,N′-bis(â-alanyl)-1,4-diaminobenzene as a
spacer yielded a biologically active peptidomimetic analog 4
(Scheme 1). Molecular modeling experiments (data not shown)
showed that low energy conformations of this spacer would be
able to accommodate either the “parallel” or “antiparallel”
pharmacophore model (Figure 3).
Acknowledgment. We wish to thank Physical and Structural
Chemistry for mass spectral support and Robert Sanchez for amino
acid analyses of the final products.
Supporting Information Available: Experimental details and
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JA962919H