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. Author manuscript; available in PMC: 2012 Jul 6.
Published in final edited form as: J Am Chem Soc. 2011 Jun 15;133(26):10184–10194. doi: 10.1021/ja201878v

Design, Synthesis, and Validation of a β-Turn Mimetic Library Targeting Protein–Protein and Peptide–Receptor Interactions

Landon R Whitby †, Yoshio Ando †, Vincent Setola ‡, Peter K Vogt †, Bryan L Roth ‡, Dale L Boger †,*
PMCID: PMC3134394  NIHMSID: NIHMS303387  PMID: 21609016

Abstract

The design and synthesis of a β-turn mimetic library as a key component of a small molecule library targeting the major recognition motifs involved in protein–protein interactions is described. Analysis of a geometric characterization of 10,245 β-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a β-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities (Ki = 390 nM and 23 nM, respectively) for the κ-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4,200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Unique to the design and because of the C2 symmetry of the template, a typical 20 × 20 × 20-mix (8,000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid–liquid or liquid–solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands, but also additional side chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as Ki = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-Pro-Trp/Phe-Phe-NH2 β-turn is important for MOR binding, but may not be important for KOR (accommodated, but not preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a chlorine substituent further enhancing KOR affinity.

Introduction

The interaction of proteins to form higher order complexes is critical to nearly all biological processes, including cellular signaling.1 Despite their central role in cellular signaling, such protein–protein interactions have emerged relatively slowly as viable small molecule therapeutic targets.2 This can be attributed to the fact that protein–protein interfaces do not typically bind endogenous small molecule ligands that could provide lead structures for drug discovery programs and that the interfaces often also present physical challenges to small molecule binding. These challenges are generally thought to come in the form of the noncontiguous binding regions within the interacting proteins as well as an often relatively large and/or flat binding interface. However, systematic case studies of selected protein–protein interactions have revealed that there is typically a small cluster of key residues near the center of the interface that contributes the majority of the recognition or binding affinity.3 Significantly and although endogenous small molecule ligands may not exist, many if not most protein–protein interactions are mediated by three main recognition motifs (α-helix, β-turn, β-strand).4 Consequently, an attractive approach for the discovery of modulators of protein–protein interactions is to mimic the key interaction residues using small molecule mimetics of these three major recognition motifs.

A related class of interactions in which small molecule mimetics of peptide secondary structure have already proven valuable is the peptide–receptor interactions, the best characterized example of which is the interaction of the peptide-activated G protein-coupled receptors (GPCRs) with their endogenous peptide ligands. Significantly, GPCRs comprise the targets of over one-third of the currently registered pharmaceuticals, although only a subset of GPCRs are peptide-activated. Although the peptide ligands of this subset of GPCRs are usually flexible and able to adopt variable secondary structures, the active conformation recognized by the receptor typically involves a turn structure (β- or γ-turn).5 Due to this pervasive recognition pattern, small molecule mimetics of turn structures (typically β-turn)5 and peptides with turn constraints have been successful in targeting this class of receptors.5,6 As the roles of these receptors have emerged, small molecule scaffolds capable of mimicking peptide turn structures continue to be valuable in defining the residues and secondary structure responsible for the binding recognition and affinity, useful in validating new drug targets, and central to the subsequent development of new therapeutics.6b

Over the past decade, we have enlisted a simple solution-phase library synthesis protocol, complementary to more conventional solid-phase techniques, for the generation of libraries capable of targeting protein–protein or protein–DNA interactions.7 The protocol employs acid/base liquid–liquid or liquid–solid extractions for the purification of products (>95% pure irrespective of the reaction efficiency), and offers the advantages of a less limiting scale, expanded repertoire of chemical reactions (use of heterogeneous catalysts and reagents), direct production of soluble intermediates and final products for assay, and the lack of required linking, attachment and detachment, or capping steps. The approach is amenable to convergent synthetic strategies, the synthesis of mixture libraries, or the use of dynamic libraries. Notably, a number of effective small molecule modulators of protein–protein7–12 or protein–DNA interactions13 have been identified from screening the libraries prepared to date.

Recently, we initiated a program to expand our current library of 95,000 compounds2f,7 with the preparation of a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate protein–protein interactions, namely the α-helix, the β-turn, and the β-strand. Three libraries built around templates designed to mimic each secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains would contain a member capable of mimicking the key interaction residues of many, if not most, protein–protein interactions.4 Such a library would provide a powerful tool to interrogate protein–protein interaction networks in order to validate new therapeutic targets, to provide therapeutic lead compounds, and to afford modulators of biological processes for study. The screening of the comprehensive library would not only provide lead structures for many protein–protein interaction targets even if the nature of the interaction is unknown, but it can also be expected to yield key insights into the recognition motif and key residues mediating the interaction.14 Screening of the library would also provide an initial comprehensive structure–activity relationship (SAR) study for subsequent iterative lead optimization.14 Notably, the β-turn component of this library would be particularly valuable for the protein- and peptide-activated GPCRs that recognize turn structures in their endogenous ligands.4,5 To date we have disclosed the design, synthesis and validation of an 8,000-membered α-helix mimetic component of this library15 and we disclose herein the second component of this comprehensive library, a β-turn mimetic library targeting protein–protein and peptide–receptor interactions.

Results and Discussion

β-turn mimetic design

The β-turn is one of the three main secondary structural motifs found in proteins and peptides and occurs where the polypeptide strand reverses direction. As illustrated in Figure 1, the β-turn consists of four amino acid residues designated i to i+4 in which the distance between Cαi and Cαi+3 is 7 Å. Several different types of β-turns exist, depending on the dihedral angles ψ and ϕ of the i+1 and i+2 residues.

Figure 1.

Figure 1

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Schematic diagram of a β-turn.

Small molecule mimetics of β-turns have been extensively investigated and utilized to discover compounds that can mimic or disrupt β-turn mediated recognition events.16,17 An ideal β-turn mimetic scaffold around which to build a screening library would be constrained to approximate the correct geometric display of the amino acid side chain functionality found within a β-turn, be sufficiently flexible to allow the side chains to approximate the side chain vectors of the many turn types, and be amenable to robust library synthesis.

We envisioned a β-turn mimetic scaffold that could be substituted with all triplet combinations of groups representing the 20 natural amino acid side chains that was amenable to substitution using our solution-phase library synthesis protocol. This would enable production of a library of pure compounds (>95%) on a scale that would permit virtually unlimited screening opportunities and enable efficient optimization of lead compounds through the rapid synthesis of subsequent lead optimization libraries. In order to identify a template that would allow the appropriate geometric display of the amino acid side chain functionality, we utilized a geometric analysis of the mean distances found between α-carbon centers in a set of 10,245 β-turns in the protein data bank (PDB, Figure 2).18,19

Figure 2.

Figure 2

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Mean and standard deviations of the Cα distances taken from a set of 10,245 β-turns in the PDB.

Recognizing that one of the turn amino acids (i+1 or i+2) often serves a structural rather than a recognition role (e.g., Pro or Gly),4,18 we sought to mimic Cα triplets in which either Cαi+1 or Cαi+2 is omitted (i.e., Cαi, Cαi+2, Cαi+3 or Cαi, Cαi+1, Cαi+3). From this analysis, we found that trans-pyrrolidine-3,4-dicarboxamide could serve as a synthetically accessible template upon which to display the amino acid side chain groups (Figure 3A). Rigidified by an intramolecular H-bond, the lowest energy conformation of the trans-pyrrolidine-3,4-dicarboxamide has substituted centers that conform closely to the triangle geometries of the Cα triplets found in Figure 2 in which either Cαi+1 or Cαi+2 is omitted (Figure 3B). An overlay of the low energy conformation with the peptide backbone of a type I β-turn (Figure 4A and 4B) demonstrates a potential mode of mimicry and yielded an rmsd value of 0.26 Å for ethyl substituted centers versus the Cα triplet Cαi, Cαi+2, Cαi+3. In line with the design, the non-covalent constraints that stabilize the lowest energy conformer also permit a degree of flexibility to allow the compound to adopt variable H-bond donor/acceptor patterns (which carbonyl is exo or endo in pseudo 7-member ring) and permit the attached side chains to approximate the correct vector display of amino acid side chains in a wide range of β-turn structures.18 Significantly, the trans-pyrrolidine-3,4-dicarboxamide can be substituted using amide coupling reactions and possesses a simplifying C2 symmetry axis that allows all triplet combinations of 20 different side chains to be accomplished with only 4,200 compounds (versus 8,000). Thus, in addition to potentially serving as a -turn mimetic accurately matching the triangle geometries of the Cα triplets of a -turn while flexibly accommodating the vector displays of side chains in a wide range of β-turn structures,18 it also possesses a unique simplifying C2 symmetry and is functionalized in a manner amenable to library synthesis.

Figure 3.

Figure 3

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(A) The trans-pyrrolidine-3,4-dicarboxamide template. (B) Low energy conformation and measured distances between substituted centers of a trans-pyrrolidine-3,4- dicarboxamide with ethyl substituents. (C) A trans-pyrrolidine-3,4-dicarboxamide substituted with groups representing the side chains of Leu, Phe, and His.

Figure 4.

Figure 4

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(A) Overlay of a potential mode of β-turn mimicry by a trisubstituted trans-pyrrolidine-3,4-dicarboxamide. (B) Overlay and calculated rmsd value for attachment of the three substituted centers of the template with three α-carbons in a type I β-turn.

Evaluation and validation of the design against peptide-activated GPCRs: the opioid receptors

In order to establish the ability of substituted trans-pyrrolidine-3,4-dicarboxamides to modulate βturn mediated recognition events and before embarking on a comprehensive library synthesis, the evaluation of a select series of compounds against a representative set of peptide-activated GPCRs that are thought to recognize β-turns in their endogenous ligands was conducted. The μ-opioid receptor (MOR) is a well known clinical target for the treatment of pain. MOR selective opiate analgesics such as morphine remain the drugs of choice for the treatment of severe pain, but their use is limited by the well-characterized side effects of respiratory depression, desensitization with chronic use (tolerance), and development of dependence. The additional two members of the opioid receptor family, the κ-opioid and δ-opioid receptors (KOR and DOR), have also been investigated as analgesic targets. DOR agonists produce analgesia, but may also exhibit side effects including convulsions. Activation of the KOR in the central nervous system produces analgesia, though it is generally accompanied by dysphoria, hallucinations, and sedation. Targeting of peripheral KOR, however, has emerged as a potentially promising treatment for inflammatory and visceral pain as well as arthritis.20

Two highly potent and selective endogenous peptide ligands of the MOR are endomorphin-1 (H-Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2).21 Additional endogenous agonists include Met-enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) and Leu-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) that show high affinity for MOR and DOR, their longer peptide precursors β-endorphin and dynorphin, as well as their C-terminus truncated neoendorphin peptides that display high affinity for the KOR.20 Although some uncertainty remains, there is considerable evidence that the active conformation of the endomorphins is a β-turn, including activities observed by analogues that incorporate turn constraints and appropriately substituted β-turn mimetics.22 Therefore, we prepared a small series of pyrrolidine-3,4-dicarboxamide derived compounds designed to mimic the three side chain residues in the pharmacophores of the endomorphins and enkephalins along with several Ala negative controls (17–22) and measured their binding (Ki values) to MOR, KOR and DOR (Figure 5). The highest affinity for the MOR was exhibited by compound 10 with the R1/R2 combination of Trp/HoPhe and R3 of Tyr (Ki = 820 nM) followed by compound 8 with R1/R2 of Trp/Phe and R3 of HoTyr (Ki = 930 nM). In addition to exhibiting submicromolar activity using a simple template, the activity reflected a combination of side chain residues found in the endogenous endorphin ligands. Moreover, comparisons among the compounds examined demonstrated that the best affinity to MOR was observed when single carbon extensions of key side chains (HoPhe or HoTyr) were incorporated into the structure and this influenced our selection of side chains incorporated into the full library design. Interestingly, the compounds showed much higher affinities for the KOR than for the MOR, highlighted by 8 and 9 with Ki values measured at 390 nM and 23 nM, respectively (Figure 6). The identification of 9 as the most active compound is easy to appreciate considering that the R3 Tyr side chain is attached to the pyrrolidine nitrogen via an alkyl rather than acyl linkage giving rise to the familiar tyramine group bearing a free amine found in the “message” structure of nearly all opioid analgesics and peptides (Figure 6B).20a However, even its N-acyl variant lacking the basic nitrogen (8, Ki = 390 nM) proved remarkably effective. Selectivity for KOR binding was observed with 9 (~100 fold) and this trend was observed with nearly all compounds tested. The origin of this selectivity is currently undefined, but its consistency across the range of compounds tested suggests that there are intrinsic properties to the trans-pyrrolidine-3,4-dicarboxamide template, including its 3-dimensional projection of side chains or interactions made by the template itself, that favor KOR binding. Nevertheless, the high nanomolar affinities of compounds 8 and 10 indicate that the trans-pyrrolidine-3,4-dicarboxamide template can effectively mimic the active turn structure of the endomorphins.

Figure 5.

Figure 5

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Screening data of compounds against μ-opioid receptor (MOR) and κ-opioid receptor (KOR).

Figure 6.

Figure 6

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(A, B) Structures of high affinity, KOR selective compounds 8 and 9. The tyramine pharmacophore found in the “message” structure of opioid compounds and peptides is highlighted in B.

Synthesis of the β-turn mimetic library

Convinced that the pyrrolidine-3,4-dicarboxamide template would prove to be a valuable β-turn mimetic and having established subtle design features from the exploratory studies, the synthesis of the comprehensive β-turn mimetic library was initiated. The 20 amino acid side chain groups utilized to construct the library along with corresponding protecting groups are provided in Figure 7. By substituting each position of the trans-pyrrolidine-3,4-dicarboxamide template with all combinations of these 20 groups, a total of 4,200 compounds are produced representing all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Due to the perceived degeneracy of incorporating the side chains of both aspartic and glutamic acid as well as asparigine and glutamine, only the side chains of glutamic acid (Glu) and glutamine (Gln) were used in the library. Similarly, an arginine side chain was omitted, but the side chain of lysine (Lys) was incorporated. No attempt to incorporate a cysteine side chain was made due to anticipated stability and storage problems. Finally, the side chains of glycine and proline were omitted due to their expected minor role in recognition events. These six natural amino acid side chain omissions were replaced with additional unnatural aromatic side chain groups that often dominate protein–protein and protein–peptide interactions or with groups that represent one carbon extensions of such residues. Thus, O-methyl tyrosine (TyrMe), naphthyl (Nap), and 4-chlorophenylalanine [Phe(4Cl)] were included in the library as well as the side chains of the one carbon extension residues homophenylalanine (HoPhe), homotyrosine (HoTyr) and 4-chlorohomophenylalanine [HoPhe(4Cl)]. Having selected the 20 groups to incorporate into the library, they were used in their terminal amine form (for incorporation into the R1/R2 positions) or terminal carboxylic acid form (for incorporation into the R3 position) for the library synthesis.

Figure 7.

Figure 7

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The 20 groups used in the synthesis of the β-turn mimetic library.

The retrosynthetic analysis for the construction of the library is shown in Scheme 1. We anticipated that the final 4,200 compound library would be obtained by acylation of 210 individual trans-pyrrolidine-3,4-dicarboxamides at the pyrrolidine nitrogen using an equimolar mixture of the carboxylic acids of the 20 selected side chains (210 × 20-mix). The order of the side chain introductions (amide couplings; R1, R2, and R3) and the location of the 20-mix functionalization (R3) were dictated by the C2 symmetry of the template and the simplifying opportunity it presented for the number of compounds required to represent all 20 × 20 × 20-mix combinations (210 individual R1/R2 combinations x 20-mix for R3). The 210 individual amines were anticipated to be derived from their 2,2,2-trichloroethoxycarbonyl (Troc) protected precursors, which were individually synthesized starting from the mono methyl ester of trans-N-Troc-pyrrolidine-3,4-dicarboxylic acid utilizing a straightforward amide coupling, methyl ester hydrolysis, and amide coupling reaction sequence. In turn, the mono methyl ester was to be prepared from the N-benzylpyrrolidine mixed benzyl methyl ester using hydrogenolysis of the benzyl group followed by Troc protection of the free amine. Finally, the trans-N-benzylpyrrolidine-3,4-dicarboxylic acid benzyl methyl ester could be accessed using a [3+2] dipolar cycloaddition between the mixed fumarate ester and the azomethine ylide precursor N-benzyl-N-methoxymethyl-N-(trimethylsilyl)methylamine.23

Scheme 1.

Scheme 1

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Retrosynthetic plan for construction of the β-turn mimetic library.

The synthesis of the mono methyl ester of trans-N-Troc-pyrrolidine-3,4-dicarboxylic acid (26) is shown in Scheme 2. The azomethine ylide precursor N-benzyl-N-methoxymethyl-N-(trimethylsilyl)methylamine (23) was prepared as described23 by condensing benzylamine with chloromethyltrimethylsilane to yield N-benzyl-N-(trimethylsilyl)methylamine, followed by treatment with formaldehyde/H2O/MeOH in the presence of K2CO3. This material was employed in a [3+2] dipolar cycloaddition with the mixed fumarate ester in the presence of LiF and under ultrasound conditions to give 24 in multigram quantities.23 This material was further elaborated to the amino acid 25 by hydrogenolysis removal of the benzyl groups [H2, Pd(OH)2] followed by Troc protection of the pyrrolidine nitrogen using 2,2,2-trichloroethyl chloroformate to provide 26.

Scheme 2.

Scheme 2

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With the starting template in hand, the library diversification began with the first amide coupling using 20 primary amines under EDCI/HOAt mediated coupling conditions to provide the monoamides (eq 1 and Figure 8), which were fully characterized by 1H and 13C NMR, IR, and HRMS (Supporting Information). Methyl ester saponification (LiOH) provided the individual 20 monosubstituted carboxylic acids in high yields.

Figure 8.

Figure 8

Figure 8

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Synthesized 20 monoamides with coupling yields and product amounts.

graphic file with name nihms303387e1.jpg (1)

The next stage in the library synthesis entailed preparation of the 210 trans-pyrrolidine-3,4-dicarboxamides (eq 2). The Troc-protected dicarboxamides were prepared by amide coupling (EDCI, HOAt, 2,6-lutidine, DMF, 25 °C, 14 h) and this enabled isolation and purification of the products (62–100% yield) using acid/base liquid–liquid extractions in which all reagents, reagent byproducts, and any unreacted starting materials were removed. A diagonal set of 20 diamides of the full 210 matrix, representing a member of each of the monamides and each of the R2 amine coupling reactions, was fully characterized (1H and 13C NMR, IR, and HRMS) confirming both the compound structure and purity (>95%) (Supporting Information). The subsequent Troc deprotection was conducted using activated zinc nanopowder (20 equiv) in 2:1 THF/AcOH (25 °C, 8 h).24 Following zinc treatment, the compounds were isolated by filtration through Celite to remove the zinc and removal of the solvent in vacuo with a toluene azeotrope. Residual AcOH was completely removed by dissolving the compound in MeOH and passing it through a small column of basic silica gel (Chromatorex NH, Fuji Silysia Ltd). This procedure provided highly pure amines (> 95%) in good yields (product yields and amounts in Supporting Information) and avoided the use of an aqueous base extraction, which was found to result in low product recovery in certain cases due to the aqueous solubility. As before, a representative but different diagonal set of 20 compounds of the full 210 matrix was fully characterized by 1H and 13C NMR, IR, and HRMS (Supporting Information).

graphic file with name nihms303387e2.jpg (2)

The final library was obtained by coupling (EDCI, HOAt, 2,6-lutidine, DMF, 25 °C, 12 h) each of the 210 amines with an equimolar mixture of the carboxylic acids of the 20 groups shown in Figure 10 (eq 3). To ensure that each of the 20 carboxylic acids was fully consumed to yield an equimolar mixture of 20 products, 1.2 equivalents of the free amine was employed in each coupling reaction. The excess starting amine was subsequently removed upon completion of the reaction using aqueous acid extractions. The final global deprotection of all side chain protecting groups was accomplished by treatment with 3:1:1 TFA/MeOH/H2O (25 °C). Using this condition, complete removal of Boc, tert-butyl ester, TIPS, and trityl groups was observed in 12–16 h. We then verified that all final products in 20 representative final mixtures were detected by MS using a third diagonal 20/210 matrix characterization (see Supporting Information).

Figure 10.

Figure 10

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(A) % inhibition of [3H]U69593 binding for the indicated R1/R2 side chain combinations in the screening of the 210 mixtures against the KOR. (B) % inhibition of [3H]DAMGO binding for the indicated R1/R2 side chain combinations in the screening of the 210 mixtures against the MOR.

graphic file with name nihms303387e3.jpg (3)

To confirm the quality of the construction of the library as 20 compound mixtures, a representative final library mixture was selected for comparison by HPLC to an authentic equimolar mixture prepared from the individually synthesized compounds (Figure 9). Although a separation of all 20 mixture components is not possible on a single HPLC run, the nearly identical detection profiles displayed by the two mixtures confirms that not only are all 20 compounds present in the library mixture, but that they must be present in amounts that approach equimolar.

Figure 9.

Figure 9

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HPLC comparison of library (EXP 1) and authentic (EXP 2) equimolar mixture of final 20 compounds derived from 27. HPLC conditions: linear gradient 0–90% MeCN in H2O over 7 min, then 90% MeCN for 10 min at flow rate of 0.75 mL/min.

Screening the library against the opioid receptors

The entire library composed of 210 mixtures of 20 compounds (210 wells) was screened at 10 μM (total concentration, 0.5 μM per compound) for activity at opioid receptors in radioligand binding assays at human cloned receptors (KOR, MOR, and DOR). The summary of the screening results for the KOR and MOR is shown in Figure 10. The overall trends against the three receptors proved consistent with our previous single compound results in exhibiting an intrinsic binding selectivity of the trans-pyrrolidine-3,4-dicarboxamides for the KOR, followed by the MOR and then DOR (data not shown). A clear SAR trend is evident from the primary screening results even without the examination of individual compounds, illustrating that hydrophobic aromatic side chains generally dominate the interaction with the opioid receptors, with Phe(4Cl), Trp, and Nap showing strong inhibition against the KOR and the HoTyr, Trp, and HoPhe(4Cl) side chains ranking among the best against the MOR. The exception to this trend with both the KOR and MOR was the potent inhibition observed with a basic His side chain in the R1/R2 position, with a particularly potent R1/R2 combination being His and a bulky aliphatic residue (Val or Leu).

Deconvolution of selected mixtures and screening of individual compounds

Based on the primary screening data for the library and with the recognition β-turn sequence of the endomorphins (H-Tyr-Pro-Trp/Phe-Phe-NH2) in mind, three related mixtures were selected for deconvolution (Figure 11). The series containing R1/R2 side chains Trp and Phe is the closest representative within the library of the endomorphin-1 sequence and contains compound 8, which exhibited the measured Ki values of 390 nM and 930 nM for KOR and MOR, respectively. The Trp/Phe(4Cl) series represents a closely related series to the Trp/Phe series, but the addition of the 4-Cl substituent enhanced binding of the mixture to both the KOR (86% vs 73% inhibition) and the MOR (66% vs 56% inhibition). Its deconvolution could be expected to yield individual compounds with enhanced affinity to both receptors while also allowing a further evaluation of the β-turn mimicry of the compounds. The final series chosen for deconvolution, the Phe(4Cl)/Phe(4Cl) series, was the mixture that displayed the highest affinity against the KOR (91% inhibition) and also the greatest difference between KOR and MOR affinity (91% vs 48%). These three mixtures were deconvoluted by resynthesis of the individual compounds from the three archived penultimate intermediates using the conditions found in equation 3 to yield 60 individual compounds for screening.

Figure 11.

Figure 11

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Mixtures selected for deconvolution.

The 60 individual compounds were screened at 10 μM for activity at the KOR and MOR via the radioligand binding assays. Because of the lower activity at the MOR, where the % inhibition was not pegged at levels approaching 90% for each series, the general trends were clearest in examining its results. The R3 side chain preferences for the individual compounds against the MOR are shown below (Figure 12). According to the proposed mode of β-turn mimicry by the compounds and the sequence of the MOR selective endomorphins, Tyr or HoTyr would be expected to be the favored side chain at the R3 position if mimicry of an endomorphin β-turn is being achieved. Consistent with this expectation, the screening demonstrated that either HoTyr or Tyr was the favored R3 substituent in all three series, with HoTyr performing best in the Trp/Phe and Trp/Phe(4Cl) series and Tyr yielding the most potent compound in the Phe(4Cl)/Phe(4Cl) series. Beautifully, and if the turn recognition motif were unknown, these results would represent the identification of the Tyr-XXX-Trp-Phe and Tyr-XXX-Phe-Phe β-turn motifs. This result also reinforced our earlier decision to incorporate singe carbon extensions of key side chains such as Tyr into the library. In general, other aromatic R3 side chains were less active but roughly equal in potency and all were typically more active than compounds bearing the aliphatic or charged side chains regardless of the series.

Figure 12.

Figure 12

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Screening results (% inhibition at 10 μM) for the three series of deconvolution compounds against MOR along with measured Ki values (nm) (vs [3H]DAMGO).

The data from each of the three deconvolution series is shown in Figure 13. Clear from these comparisons is the greatest activity against the KOR (KOR > MOR > DOR) for all three series and that the activity of the three series generally follows the order of Phe(4Cl)/Phe(4Cl) > Trp/Phe(4Cl) > Trp/Phe as observed in the original mixture screening results (Figure 11). The excellent activity of 8 (Ki = 390 nM) used originally to test the design and found in the Trp/Phe series was improved with the replacement of Phe with Phe(4Cl) in the Trp/Phe(4Cl) series providing 28 (Ki = 250 nM, Figure 14A) and this series provided several related compounds that exhibited Ki’s of < 300 nM. Finally, the most active Phe(4Cl)/Phe(4Cl) series not only showed a greater activity for the KOR, but it also exhibited a greater selectivity for the KOR versus the MOR or DOR as inferred from the original mixture screening results (Figure 11). The most potent compound in this series for the KOR was 29 (Ki = 80 nM, Figure 17B) that was found to be >100-fold selective versus the MOR or DOR (Ki >10,000 nM).

Figure 13.

Figure 13

Figure 13

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Screening results (% inhibition at 10 μM) for the three series of deconvolution compounds against KOR (vs [3H]U69593), MOR (vs [3H]DAMGO), and DOR (vs [3H]DADLE) along with measured Ki values (nM) against KOR.

Figure 14.

Figure 14

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Chemical structures of compounds with highest affinity for KOR from the Trp/Phe(4Cl) series (28) and the Phe(4Cl)/Phe(4Cl) series (29).

As detailed earlier, the trends for MOR binding illustrated that the HoTyr [Trp/Phe, Trp/Phe(4Cl)] or Tyr [Phe(4Cl)/Phe(4Cl)] were the clearly favored R3 substituent in all three series indicative of a prominent or productive role for the Tyr free phenol. Other aromatic R3 side chains were less active and roughly equal in potency (Figure 12). This unique preference for HoTyr or Tyr and the presence of the free phenol was not observed with KOR. While still preferring a third aryl R3 side chain substituent [Phe(4Cl), HoTyr > Tyr = Tyr(Me), Phe > HoPhe(4Cl), > Trp, HoPhe > Nap] and accommodating the free phenol of Tyr, it no longer exhibits a phenol preference, Figure 15. Without over interpreting the minor differences in the measured and sometimes pegged assay values, there was virtually no difference in Tyr and Tyr(Me) for each of the three series indicating that protection of the phenol as a methyl ether has no impact in binding affinity, and both were nearly identical to Phe itself in each of the three series. Among the best of the R3 substituents in nearly each of the three series was Phe(4Cl) and HoPhe(4Cl). In addition to suggesting that the Tyr free phenol of the endomorphins and enkephalins may not be important to KOR binding,25 it also suggests that a way to enhance KOR versus MOR and DOR binding selectivity is to remove this pharmacophore phenol OH. Like the behavior of 29, further enhancements in KOR affinity may be achieved by utilizing Phe(4Cl) resulting in higher affinity and even more selective KOR ligands.

Figure 15.

Figure 15

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Screening results (% inhibition at 10 μM) for the three series of deconvolution compounds against KOR (vs [3H]U69593).

Conclusions

The solution-phase synthesis of a β-turn mimetic library as the second component of a general small molecule library targeting the key recognition motifs involved in protein–protein interactions is described. Using a geometric characterization of β-turns in the PDB, the trans-pyrrolidine-3,4-dicarboxamide was proposed and subsequently found to serve as an effective and synthetically accessible library β-turn mimetic template and this was initially validated by screening test compounds against a series of peptide-activated GPCRs that recognize β-turn structure in their ligands. The screening of selected compounds designed to mimic the β-turn pharmacophore of the peptide ligands of the opioid receptors demonstrated that appropriately substituted trans-pyrrolidine-3,4-dicarboxamides could replace the peptide backbone for effective display of side chain groups in a β-turn. This validation was highlighted by identification of both nonbasic and basic small molecules with high affinity (Ki = 390 nM and 23 nM, respectively) for the κ-opioid receptor (KOR). The library was assembled using a solution-phase protocol to provide 210 mixtures of 20 compounds for a total of 4,200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Even if the recognition motif is unknown or unrecognized, the library screening should provide lead structures, provide insights into the nature of the interaction (β-turn), and identify the key amino acid residues responsible for the protein–protein or peptide–receptor interaction. Additionally, the use of such a comprehensive library can take advantage of the principles of selection to discover compounds with improved properties (e.g., of affinity, selectivity) over those that mimic the endogenous protein or peptide ligands. The screening of the complete library against the opioid receptors demonstrated that not only could the expected activity be observed with library mixtures containing the compounds shown to mimic the opioid receptor endogenous peptide ligands, but that additional side chain combinations could be identified with even higher receptor binding affinities and selectivity providing new insights into the -turn recognition events.

Supplementary Material

1_si_001

Acknowledgments

We gratefully acknowledge the financial support of the National Institutes of Health (CA078045, DLB and PKV; R01DA017204, R01DA027170, the Michael Hooker Distinguished Professorship and the NIMH Psychoactive Drug Screening Program Contract, BLR). LRW is a Skaggs Fellow.

Footnotes

Supporting Information Available. Full experimental details on GPCR screening conditions and the library synthesis along with single compound characterizations of all 20 monoamides (eq 1: 1H and 13C NMR, IR, and HRMS), a diagonal set of 20 Troc-diamides of the full 210 matrix representing a member of each of the monamides and each of the R2 amine coupling reactions (eq 2: 1H and 13C NMR, IR, and HRMS) confirming the compound structure and purity (>95%), a table summarizing the LCMS characterization of an additional 50 Troc-diamides establishing their isolation (MS) and purity, compound characterizations of a second but different diagonal set of 20 compounds in the full 210 matrix of diamide amines (eq 2: 1H and 13C NMR, IR, and HRMS), MS characterization of a third diagonal set of 20 of the final 210 mixtures confirming the presence of all 20 components, the characterization of the 33 individual final triamides constituting deconvolution samples (1H NMR, MS), and three tables summarizing the isolated amounts and yields of each component in the three stages of the library synthesis are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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Supplementary Materials

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