Non-Peptidic Bradykinin Receptor Antagonists From a
Structurally Directed Non-Peptide Library
Sarvajit Chakravarty, Babu J. Mavunkel, Robin Andy, Donald J. Kyle*
Scios Nova Inc.
820 W. Maude Avenue
Sunnyvale, California 94086
In most mammals, "kinins" refer to the nonapeptide bradykinin (Arg1-Pro2-Pro3-Gly4- Phe5-Ser6-Pro7-Phe8- Arg9) and the decapeptide kallidin (Lys1- Arg2-Pro3-Pro4-Gly5- Phe6-Ser7-Pro8-Phe9- Arg10). In rats another kinin (T-kinin) is produced under certain circumstances and binds to the same receptors as bradykinin . A schematic of the human kinin-kallikrein system is shown in Figure 1
The release of kinins from precursor proteins (known as kininogens) is mediated by enzymes called kininogenases [2,3]. The predominant enzymes responsible are kallikreins, but others, which include trypsin, plasmin, and some snake venoms also release kinins. Kininogens are primarily synthesized in the liver and represent an abundant source of the precursors which are required for kinin generation. Kininogen-like substances are also found associated with several cell types, suggesting that kininogen production may be a local event . These proteins are produced from alternative splicing of a single gene product. There are two forms, high molecular weight kininogen (HMWK), and low molecular weight kininogen (LMWK).5 Unlike HMWK which exists in the circulation as a complex with plasma pre-kallikrein, LMWK circulates freely.
During immunological reaction, charged surfaces, which may be derived from bacterial lipopolysaccharide, oligosaccharides, connective tissue proteoglycans or damaged basement membranes, enable factor XIIa. Once factor XIIa is present, pre-kallikrein can be cleaved to its active form, known as plasma kallikrein. This enzyme acts upon its preferred substrate, HMWK, to release the nonapeptide bradykinin. Plasma kallikrein is further able to convert inactive factor XII to active XIIa, thereby participating in a positive feedback loop. The cleavage of bradykinin from HMWK is highly localized since pre-kallikrein and substrate (HMWK) circulate as a complex.
Another kinin, Lys-bradykinin (also known as kallidin), is produced via the action of an enzyme named tissue kallikrein on LMWK. This enzyme is found in many tissues, either in the form of a precursor requiring activation or as an active enzyme. In contrast to plasma kallikrein which preferentially acts upon HMWK, tissue kallikrein can release kallidin from either HMWK or LMWK. Through the action of aminopeptidases, kallidin can subsequently be converted directly into bradykinin. This enzyme is present in both the plasma and on the surface of epithelial cells.
Both bradykinin and kallidin can be degraded by a variety of plasma and cell surface enzymes (kininases) . The most widely recognized of these enzymes are kininase I, kininase II (angiotensin converting enzyme, ACE), and carboxypeptidase N. In plasma, kininase I cleaves the C-terminal arginine from both bradykinin and kallidin to form [des-Arg9] kinins. These [des- Arg9] kinins are known to act as agonists of B1 receptors which are present in some species and have been implicated in the pathophysiology associated with prolonged inflammation [7-9].
Nearly all cells express kinin receptors which mediate the activities of both bradykinin and kallidin. The activation of these G-protein coupled receptors causes relaxation of venular smooth muscle and hypotension, increased vascular permeability, contraction of smooth muscle of the gut and airway leading to increased airway resistance, stimulation of sensory neurons, alteration of ion secretion of epithelial cells, production of nitric oxide, release of cytokines from leukocytes and eicosanoids from various cell types. Because of this broad spectrum of activity, kinins have been implicated in many pathophysiologies including pain, sepsis, asthma, symptoms associated with rhinoviral infection, rheumatoid arthritis, and a wide variety of other inflammatory diseases. A bradykinin antagonist used clinically to treat any of these disorders would represent a completely novel therapy.
Depending on the available structural information for a given target protein and/or a collection of ligands which bind to it, there are different structure-based design strategies which can be considered. In a broad sense, there are two major categories for these strategies which are, (1) those based on receptor binding site structure and (2) those based on the bio-active conformation(s) of known ligands. The former can rely on detailed information such as that obtained from x-ray crystallographic data or NMR experiments, or the method may rely on structural information deduced from the results of mutagenesis experiments. The latter category is generally applied to systems where little or no structural information regarding the ligand binding site is known. The approach is to introduce conformational constraints into a series of ligands then determine their respective affinities for a given target protein. Frequently, this approach is prefaced by an investigation of the solution conformation of the native ligand.
We have taken a structure-based approach to the design and synthesis of non-peptide bradykinin antagonists. Since no explicit structural data is available for the ligand binding site(s), agonist and antagonist binding sites have been elucidated using molecular biology tools (i.e. receptor chimeras, mutagenesis, etc.), computational procedures, and receptor-probing ligands. In parallel, several series of conformationally constrained ligands have been prepared which explore the requisite topological features of high affinity ligands. The elucidation of binding site structures via these indirect methods results in structures which at best should be considered "fuzzy" images. In that context, it would be unrealistic to expect one to design a specific compound with high affinity for a given site. Our approach was instead to design non-peptide building blocks which were roughly compatible with the binding site models, and consistent with the known SAR developed previously from constrained ligands. Upon each of these building blocks, chemical diversity was introduced leading to several pools of non-peptide building blocks. The number of constituents in these pools varied from three to six such that our structurally directed libraries contained not more that 36 members. This approach is in contrast to many others where claims are made to libraries of several thousand members.
1. Bradykinin B2 receptor binding
Membranes were prepared from transfected Chinese hamster ovary cells expressing the human B2 bradykinin receptor. Cells were plated and grown to confluence in 150 mm dishes. Monolayers were rinsed two times with PBS, and pelleted by centrifugation. The cell pellet (from 10 plates) was re-suspended in 5 mL of 25 mM TES (pH 6.9, 0.019% 1,10-phenanthroline) and subjected to four passes with a type A pestle of a Dounce homogenizer. After sitting on ice for 10 min., cells were broken for 10 min. at 500 xg. Membranes were pelleted by centrifugation of the supernatant at 72,000 x g for 60 min. The crude membrane pellet was re-suspended in 4 mL 25 nM TES (pH 6.9, 0.019% 1,10-phenanthroline) and aliquots were frozen in liquid nitrogen.
Binding of [3H] bradykinin was measured by incubating 20mg membrane in 2 mL binding buffer ( 25 mM TES @ pH 6.9, 0.019% 1,10- phenanthroline, 1% BSA, 0.2 mg/mL bacitracin) containing 10-10 M [3H] bradykinin and various concentrations of the test compounds for 90 min. Bound was separated from free by filtration through Whatman GF/B filters that had been pre-soaked in 0.1% polyethyleneimine. After washing with 50 mM Tris-Cl (pH 7.4), the filters were counted. 15% or less of the total radioactivity was bound and non-specific binding was less than 4% of cpm bound.
2. Synthesis of DR-R-X-Y-R molecules
All compounds of the sequence DR-R-X-Y-R, where X and Y are independently selected from the pools on non-peptide building blocks, were synthesized using standard solid phase techniques in accordance with published procedures. Building blocks were synthesized and characterized individually prior to their use in solid phase couplings. These synthetic details will be published elsewhere. All non-peptide building blocks were coupled as their active esters, generated by pre- treatment with hydroxybenzotriazole hydrate and diisopropylcarbodiimide in methylene chloride:DMF (1:1) for 30 min. at 0 degrees C prior to introduction into solid phase synthesis. The N-terminal Boc protection was removed by successive treatments with 50% TFA in methylene chloride for 1.5 and 20 min. Subsequent to washing, the free amine on the resin was neutralized by treatment with 10% diisopropylethylamine in methylene chloride.
A given pool of building blocks was simultaneously coupled in slight excess (1.2 eq) to Arg-PAM resin to generate Boc-Y-Arg-PAM. Position X was introduced in a similar fashion, leading to Boc-X-Y-Arg-PAM. After placing the N-terminal Arg residues, the libraries were of the sequence DR-R-X-Y-R. These libraries were released from the resin by treatment with anhydrous HF/10% anisole. After removal of the excess HF, the residue was washed extensively with ether and dried under vacuum. The libraries were taken up in water/methanol/acetic acid (60/30/10) then, after removal of methanol in vacuo, lyophilized to powders. FAB mass spectroscopy was used to locate ion peaks corresponding to the individual compounds in a particular library.
A modular approach to our strategy is outlined in Figure 2 where a previously described pseudopeptide lead, NPC 18325 (DR-R- (CH2)11-C(O)-R), might be converted into a non- peptide by re-assembling the three modules illustrated as nonpeptide moieties. The three modules of NPC 18325 are (1) a positively charged N-terminal piece, (2) a mid-section containing a bend or twist, and a mimetic of Phe5, (3) a C-terminal piece of appropriate hydrophobicity and structurally simulating a type II' b-turn. On the basis of the results shown in Table 1, the second module of this lead was optimized by incorporating a variety of moieties including a 5-membered ring as a proline side chain mimetic, a benzyl group as a phenylalanine mimetic, and a double bond as a structural mimetic of a cis amide bond to introduce an overall bent shape. Several other center modules were synthetically altered to better fit the known SAR . As shown in Table 1 several scaffolds were discovered which maintained high affinity to the bradykinin B2 receptor. These representative examples of non-peptide scaffolds (i.e. spirocyclics and cinnamic acids) which were inserted to replace the second module in the parent sequence (NPC 18325) are well tolerated by the receptor as indicated by their respective affinities. One of these new pseudopeptides, NPC 18521 was subsequently shown to have in vivo bradykinin antagonist activity .
The process of fragment re-assembly led ultimately to the synthesis of several general heterocyclic templates which were ideally suited for insertion either at module 2 or 3 [12,13]. In total, 4 completely unique non-peptide templates were designed and synthesized. These include 6,5-spirocycles, beta- and gamma- carbolines, phenanthridinones and "open" phenanthridinones, and cinnamic acids. Each was designed within the framework of several criterion. First, a given scaffold must closely match the known SAR and be highly compatible with the putative ligand binding site structure. Second, each scaffold must be a relatively simple synthetic target, having readily available starting material, no chiral centers and having a total synthesis of not more that 4-5 steps. Finally, each template must have a "C-terminal" carboxylate and an "N-terminal" amino group with no interfering functionality such that it could be readily used in a solid phase synthetic strategy. To rapidly explore the receptor affinities of all possible combinations of these non-peptide templates at position X and Y of the sequence DArg-Arg-X-Y- Arg, a combinatorial synthetic approach was taken.
In this study, there were four different cinnamic acids, four different carbolines (beta and gamma), three different phenanthridinones, and five different spirocyclics. The variability (bold in Figure 3) in the phenanthridinione series was that the central ring could be opened or formed and the amino group could be meta or para substituted. In the carboline series, the cyclic amino group was either at the beta or gamma position of the cyclohexenyl ring and the methylene chain bearing the "C-terminal" carboxylate could be of variable length. The spirocyclic series was also varied in the carboxylate carbon linker length, but also in the substitution on the 5-membered ring nitrogen. The cinnamic acids had two carbon chains which could be of varying length, one of which had the further possibility of containing a double bond. Rather than perform individual syntheses of all possible combinations of these templates, members of each ring type were pooled in equimolar amounts prior to incorporation into the sequence DArg-Arg-X-Y-Arg. This diversity and pooling strategy is shown in Figure 3. Since each individual member of each pool was constructed on a similar carbocyclic scaffold, the chemical environments of the N-terminal amino group and C-terminal carboxylate groups were expected to follow similar kinetic and thermodynamic controls during synthesis of the non-peptidic sequences.
Ultimately, 12 libraries of novel non-peptidic structures were synthesized following typical solid phase methodologies. Each library contained from nine to thirty-six different compounds in approximately equimolar amounts. Figure 4 schematically shows the composition of these twelve libraries. Unpurified libraries were next tested in a receptor binding assay utilizing membrane preparations from a stable cell line expressing the human B2 receptor. Each library was tested at concentrations between 1 nM and 1 mM. The ability of each library to inhibit [3H]bradykinin binding was assessed and the results are shown in Figure 5. Although this type of screening is highly qualitative, certain libraries appear in Figure 5 which show higher affinity to the receptor than other libraries. For example, library 2 appears better than library 11. The libraries were further broken down (decoded) in order to determine which compound(s) was responsible for the activity. It is important to note that breaking these libraries down to elucidate the structure of the hit(s) was feasible due to the inherently small size of each library.
Library 2, for example, was of the series DArg-Arg-PHEN-CINN-Arg, and contained 12 different structures (recall that there were originally 3 different phenanthridinones and 4 different cinnamic acids). The first deconvolution step of the approach is shown in Figure 6. Here, only the CINN position is randomized, and the PHEN moieties were specific. This led to the preparation of three new libraries of 4 compounds each. Receptor binding was again performed as before and only one of these three new sub-libraries showed activity at 1 mM. The final step in the process required to elucidate the active component(s) was to synthesize and purify each of the 4 members of this library as shown in Figure 7. Receptor binding on these four novel non-peptidic structures showed that only one of the four had affinity to the receptor. This new compound was NPC 18884 and was subsequently shown to be an antagonist in a cellular assay measuring bradykinin- stimulated IP production. Overall, there were 285 possible structures to survey due to the number of structure-based scaffolds which were prepared. This was rapidly accomplished via 19 syntheses, 19 assays, and four purifications.
Not surprisingly, NPC 18884 showed divergent potency when assayed in different species. In particular in a model of bradykinin-induced hypotension in rats and rabbits, it appears to have no activity. Likewise, it does not block bradykinin-induced contraction of the isolated guinea pig ileum. However, it binds to the human B2 receptor with a Ki = 36 nM and is a functional antagonist in transfected CHO cells stimulated by bradykinin (IP turnover). Since it is considerably smaller than previous decapeptide antagonists, it is unlikely that NPC 18884 has as many contacts with the receptor during binding. This likely amplifies the subtle structural differences which are known to exist in species homologs of the bradykinin B2 receptor.
The discovery of NPC 18884 is significant in many regards. First, it is a highly non- peptidic lead antagonist molecule which could be further modified to reduce its mass. One approach would be the incorporation of arginine mimetics at the terminal ends. Alternatively, it may be possible to remove the C-terminal arginine residue and achieve a B1 receptor antagonist. The approach is also one of the first examples of a combinatorial synthesis of non-peptide building blocks which mimic peptide structure, ultimately tested in a non-tagged, solution-phase form. Perhaps more significant is that the success described here demonstrates a possible synergy between structure-based design and combinatorial methodology. This approach has many merits, but the most significant is the application of structurally directed libraries toward target binding site structures which, for one reason or another, may not be unambiguously characterized. This method serves to aim the combinatorial syntheses in a logical direction, rather than attempt to prepare libraries of vast diversity (and numbers).
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Pseudopeptide antagonists of the sequence
Diagram of the human kinin-kallikrein system including the native
ligands for B1 and B2 receptor subtypes.
Schematic of the overall strategy for the synthetic re-construction
of peptide-based structure-activity data based on a three module
Pooling strategy and non-peptide scaffolds which were designed and
used in combinatorial syntheses. Shown in bold are the portions of
the structures which were variable within each pool.
Composition of twelve original non-peptidic libraries of the
sequence DArg-Arg-X- Y-Arg. X and Y were selected from the set of
scaffolds shown in Figure 3.
Binding assay results for 12 non-peptidic libraries. Each library
was tested at two concentrations, 1 mM and 10 nM. Results were
compared to cold bradykinin binding which was tested at two lower
concentrations, 0.1 nM and 1 nM.
Composition of the first break-down pools from library number 2.
There were three pools of 4 compounds each of the sequence shown in
Final breakdown of library 2 showing the four non-peptidic
structures which were prepared and evaluated as bradykinin
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