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. 2024 Oct 29;15(11):2060–2066. doi: 10.1021/acsmedchemlett.4c00316

Cyclic Peptide C5aR1 Antagonist Design Using Solution Conformational Analysis Derived from Residual Dipolar Couplings

Kathleen A Farley a,*, Ye Che a, Ricardo Lira a, Peter Jones b, Nikolaos Papaioannou b, Matthew Hayward a, Mark E Flanagan a, Jonathan Langille a, Sidney Liang a, Betsy S Pierce a, Gregory Ciszewski a, Paul Bonin a, Fabien Vincent a, Simeon Ramsey b, David Hepworth b
PMCID: PMC11571007  PMID: 39563814

Abstract

graphic file with name ml4c00316_0006.jpg

To gain further insight into the conformational properties of small cyclic peptides that bind to the G-protein coupled receptor C5aR1, we report here for the first time the elucidation of three peptide solution conformations using residual dipolar couplings and NMR temperature coefficients. Each of these peptides varies by at least one amino acid, adopts a different intramolecular hydrogen bonding pattern, and has a different solution conformation. The solution conformations were used in combination with a homology structure of C5aR1 as a design template for increasing the potency of peptide leads for the C5a receptor. This study provides a framework for using RDC solution conformations to guide the design of peptide mimetics that emulate the target bound state in solution to minimize the strain energy of the bound conformation and improve potency of the peptide for the target.

Keywords: NMR, Solution conformation, RDC, Temperature coefficients, Intramolecular hydrogen bond

The complement system is the part of our innate immune system consisting of a large number of plasma proteins that play a key role in our host defense against both infectious organisms and injuries to the body with an inflammatory response.1 When the complement system is activated, a cascade of events occurs that ultimately results in the cleavage of the peptide C5 to produce the 74-residue anaphylatoxin known as C5a. The C5a peptide binds to and activates the cell surface receptor C5aR1, triggering pro-inflammatory signals. If C5a is overactivated, extensive tissue damage can result for numerous conditions such as rheumatoid arthritis (RA), lupus, septic shock, and even the so-called cytokine storm associated in patients with severe COVID-19.2 For this reason, agonism/antagonism of C5a signaling has been an attractive anti-inflammatory target for many years. Research efforts have targeted small molecule, peptides/peptidomimetics, and monoclonal antibodies.2,3 Despite the ongoing interest in the C5/C5aR1 pathway and over two decades of efforts, managing high lipophilicity/high intrinsic clearance, has been a major challenge for small molecule C5aR1 drug candidates along with poor oral bioavailability and off target activity.4−6 As a result of these challenges, avacopan was the first approved orally administered inhibitor of the C5a receptor in 2021.7

In addition to traditional small molecule approaches, significant effort has been invested in the development of peptide/peptidomimetic C5aR1 antagonists as drug candidates. Early work focused on truncating the structure of C5a and eliminating its agonistic properties.8 Later work focused on the cyclic peptide PMX-53 (Figure 1) and the related linear peptide JPE1375.9,10 Both peptides displayed activity in animal models and PMX-53 progressed to the clinic for RA before development was halted.11

Figure 1.

Figure 1

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Structures of PMX-53 (left) and JPE1375 (right).

Inspired by the orally bioavailable immunosuppressive drug cyclosporin and the observation that several other ‘Rule of 5’-breaking oral drugs are macrocycles, there has been significant recent interest in oral drugs outside the classic Lipinski space.12−15 Pfizer in collaboration with others: Scott Lokey, Matt Jacobsen, and David Fairlie have also published on several N-methylated cyclic peptides that are orally bioavailable in rats.12,16,17

Based on 1) the historical challenges in producing classical small molecule C5aR1 antagonists in low-moderate lipophilicity chemical space, 2) the precedent for obtaining C5aR1 antagonist pharmacology within a small cyclic peptide, and 3) the information gained from collaborations and other work in this area, we began work to discover a potent and orally bioavailable cyclic peptide C5aR1 antagonist.17−20 At the time of this work, the crystal structure of C5aR1 with a bound antagonist had not yet been determined.21 Instead, we based our initial SAR on a homology model of C5aR1 (Figure 2) complexed with peptide 1 (Table 1).

Figure 2.

Figure 2

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Induced-fit docking of peptide 1 complexed with a homology model of human C5aR1. The homology model of human C5aR1 was determined using the δ, μ, and κ opiod receptors for ORL1 and the chemokine receptors CXCR1, CCR5, and CXCR4. The N-termini (1–34) and C-termini (318–350) for C5aR1 are believed to be disordered, and they were omitted from the model.

Table 1. Effect of Substitution on Potency and Passive Permeability for Cyclic Peptides Where an IMHB Would Be Displaceda.

graphic file with name ml4c00316_0005.jpg

Peptide R1 R2 R3 Activity Kb (nM)b RRCKc
1 - - - 36.3 2.6
2 - - - 57.5 11.1
3 Me H H NTd 4.4
4 H Me H NTd 2.6
5 H H Me >9,500 4.2
6 - H - 5.52 5.0
7 - - - 46.5 3.4
8 - - - 82.8 6.5
9 - Me - 127 2.1
10 - - - 38.9 5.5
11 - - - 54.0 0.4
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a

The residue numbers are labeled in peptide 1. For example: compound 1 is Me-Ph1-[Orn2-NδMe-Pro3-dCha4-Phe5-(4F)-Phe6].

b

C5aR1 functional assay – human whole blood oxidative burst, geomean of N = 1–10 separate experiments.

c

Absorptive passive permeability from apical to basolateral direction was examined in the RRCK assay27 with a 30 min preincubation time and is reported as the mean (10–6cm/s).

d

Not tested, compound inactive in C5aR1 binding assay (>10uM).

Previously, the nuclear Overhauser effect (NOE) solution structure of the cyclic peptide PMX-53 was determined in DMSO. In solution, PMX-53 Ac-Phe1-[Orn2-Pro3-dCha4-Trp5-Arg6] was found to have 3 intramolecular hydrogen bonds (IMHB): Arg6 NH to Pro3 C=O, Orn2 NH to dCha4 C=O, and Orn2 NH to Trp5 C=O.22 While the structure in DMSO is helpful, we were more interested in determining the conformation of a cyclic peptide in CDCl3 since this would more closely model the membrane environment of C5a we were trying to mimic.23 We also considered determining the solution conformation in an aqueous environment, but this idea was discarded since many of these peptides were only minimally soluble in water.24 The goal of this work was to determine the residual dipolar coupling (RDC) solution conformation of one (or more) C5a antagonist cyclic peptides in chloroform to use as a starting template for potency improvements in the synthesis of novel C5aR1 antagonist peptides. We determined the solution conformation(s) to identify residues in the peptide(s) that could be modified during the design process without disrupting the IMHBs and other secondary structures.

While this method of using solution conformations to guide chemical synthesis has been discussed previously,25−27 many drug discovery teams have instead focused on computational models or the structure determination of the protein/ligand complex to guide medicinal chemistry (Figure 3). While studying the protein/ligand complex has been shown to work well, it is not always available.

Figure 3.

Figure 3

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Strategies used to guide modifications for validated hit(s). Determination of the ligand solution conformation by NMR could make significant contributions to this process if it was used more frequently.

In addition, we have found that computational models are much more reliable when supplemented with experimental measurements like residual dipolar couplings (RDC). Synthesizing large peptides libraries covering every possible permutation is another strategy, but the cost and time required to synthesize large libraries make this approach less desirable. Here, our efforts focused on using a ligand solution conformation/computational model to determine SAR and synthesize smaller targeted libraries with an improved chance of delivering active ligands.

To gain further insight into the conformational properties of cyclic peptides that bind to C5a, we report here for the first time the elucidation of three peptide solution conformations using RDC values (1D) and NMR temperature coefficients (Tc) in chloroform. Each of these cyclic peptides varies by at least one amino acid, adopts a different IMHB pattern, and has a different solution conformation. Combining RDCs and temperature coefficients allows the orientation of the carbonyl to be surmised once the orientation of the NH moiety is known. Once IHMBs were determined, the 1DNH and 1DCH couplings were used to determine the conformation of the backbone. This process can be an improvement over NOE based solution conformations since identifying sufficient long-range NOE’s can be challenging for many small molecules and peptides.26 For this reason, we have focused on determining the solution conformations of three cyclic peptides using RDCs and Tc in an NMR focused strategy to improve the potency of designed peptides for the C5a receptor. For each of the three peptide conformations we studied, we measured 9–10 backbone RDCs and were able to obtain a good fit (Q < 0.2) to a single conformer allowing us to return the solution results to the medchem team in a few days.

We explored the conformational space of the three peptides using replica-exchange with solute tempering (REST) molecular dynamics simulations in a CHCl3 solvent box as described previously.23,28 The homology model of human C5aR1 was also determined (Figure 2). Peptide 1 was docked into the homology model using an induced fit docking model and displayed a backbone structure similar to the NMR-derived solution structure of peptide 2.

Internal SAR showed that from the structure of PMX-53, we were able to (1) replace the arginine residue with hydrophobic residues, (2) replace the tryptophan residue with hydrophobic residues such as Phe, (3) remove the N-acetyl residue from the side chain Phe, (4) N-methylate the ornithine Nδ residue, and (5) retain both reasonable C5aR1 antagonist activity and obtain compounds with measurable passive permeability in the RRCK model. A more comprehensive report on the SAR for this series will be reported elsewhere.

In order to apply the RDC method, the selection of a cyclic peptide for RDC solution conformer analysis was not straightforward. Many of the initial peptides that we synthesized formed trans/cis isomers at the N-Methyl position (Orn-NδMe) at an approximate ratio of 5:1; see initial hit peptide 1 in Table 1 and proton spectrum in the SI (data on other synthesized peptides that formed trans/cis isomers not shown).29 Formation of trans/cis isomers at this ratio is undesirable since 4 sets of correlations would be observed in the NMR spectra for each resonance once added to the gel used to obtain RDC measurements (anisotropic cis, anisotropic trans, isotropic cis, and isotropic trans) instead of the expected 2 sets (anisotropic and isotropic) severely increasing spectral overlap. Substitution of an azetidine ring for the proline ring (peptide 2) increased the trans/cis ratio to >10:1 at the N-Me position and yielded the desired two sets of correlations for each RDC (Table 2, with spectra shown in SI). In addition, this substitution also increased the passive permeability determined by Ralph Russ canine kidney cells (RRCK) from 2.6 to 11.1, increasing the interest in this peptide.30 For this reason, peptide 2 was chosen as the first peptide for determination of an RDC solution conformation.

Table 2. Comparison of RDC-Selected NMR Structures of Peptides 2, 6, and 10 in CDCl3 with (a) Calculated 1DCH, 1DNH, and 3JHNHa Couplingsa.

  Experimental NMR Data
 RDC-Selected Structure
Residue (Proton #)b 1DCH (Hz) in CDCl3 1DNH (Hz) in CDCl3 3JHN-Ha (Hz) in CDCl3 Δd/ΔT (ppb/K)c Calcd 1DCH (Hz) Calcd 1DNH (Hz)
Peptide 2
azetadine (7–8) 15.3 - - - 10.2 -
d-Cha (17–18) –30.4 3.7 7.3 1.4 –29.7 3.7
Phe(4-F) (21–23) –25.6 18.7 6.4 0.5 –26.4 19.8
Phe (27–28) –13.0 –2.9 7.8 1.8 –11.2 –5.0
Orn-NdMe (3–4) –12.9 13.7 8.6 3.1 –11.9 12.8
 
Peptide 6
Pro (7–8) –1.8 - - - –0.5 -
dCha (17–18) 5.8 –11.2 9.6 1.2 6.8 –10.6
Phe(4-F) (21–23) 10.8 8.3 8.8 0 11.7 5.9
β-Phe (27–28) –18.4 –10.2 8.7 2.7 –19.6 –9.5
β-Orn-NgMe (3–4) –32.9 –9.8 9.1 0.5 –32.8 –8.7
 
Peptide 10
Thr (7–8) –13.0 5.5 8.3 1.2 –13.9 4.4
dCha (17–18) –34.3 3.9 5.6 0.6 –36.0 7.8
Phe(4-F) (21–23) –40.8 11.4 8.7 2.4 –38.9 12.3
Phe (27–28) 7.9 4.7 9.8 2.3 9.2 6.5
Orn-NdMe (3–4) –16.1 19.4 8.4 1.0 –14.4 17.3
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a

The conformation with the best fit for the RDC data in this table is shown in Figure 4.

b

Atom numbering as shown in labeled structures in SI

c

Temperature coefficient of amide proton chemical shift.

Our strategy was to combine the RDCs obtained for the backbone 1J correlations with variable-temperature proton NMR studies of the backbone NH signals to determine the backbone solution conformation and the IMHBs unequivocally. Both 1JCH and 1JNH’s were collected for cyclic peptide 2 using a compressed CDCl3-swollen PMMA gel, as previously described.31 Overall, 9 RDCs (1JCH and 1JNH) were measured for the backbone residues of peptide 2 in CDCl3. The RDCs were in the range of −30.4 to +18.7 Hz as shown in Table 2. Examination of the Tc’s for peptide 2 are consistent with three NHs involved in IMHBs: the dCha4-NH, Phe(4-F)5-NH, and Phe6-NH.

The 9 RDCs for cyclic peptide 2 were fit to a family of ∼1,000 low energy conformers using MSpin software.32 Using a conformer file this large contains many similar conformers and if we were repeating this analysis today, the file would probably contain less than 100 conformers by removing any duplicate or similar structures. The conformer with the best fit had three IMHBs: dCha4-NH to Orn2 C=O, Phe5(4-F)-NH to Pro3 C=O, and Phe6-NH to Pro3 C=O, shown in Figure 4a. Figure 4b shows the conformer with the best fit to all of the RDC data. The IMHBs shown for this conformer are in good correlation with the predicted IMHBs from the three Tc’s discussed previously for this peptide. At the time the solution conformation was first determined for peptide 1, MSpin software could not simultaneously fit to RDC’s, NOEs, and 3J-couplings, so the fit was determined only using the RDC values. The measured NOEs were used to check the goodness of the RDC fit and to ensure that the alignment media did not have an unexpected effect on the solution conformation.

Figure 4.

Figure 4

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(a) Chemical structure of peptide 2 with the three IMHBs shown in red. (b) The RDC solution conformation of peptide 2 with three IMHBs highlighted. The fit had a quality factor Q = 0.120 and a correlation factor R2 = 0.9871 using MSpin software. (c) Chemical structure of peptide 6 with the two IMHBs highlighted in red. (d) The RDC solution conformation of peptide 6 with two IMHBs highlighted. The fit had a quality factor Q = 0.081 and a correlation factor R2 = 0.992. (e) Chemical structure of peptide 10 with the three IMHBs highlighted in red. (f) The RDC solution conformation of peptide 10 with three IMHBs highlighted. The fit had a quality factor Q = 0.096 and a correlation factor R2 = 0.991. The HN spectra, HC spectra, and the experimental versus calculated backbone 1DCH and 1DNH graphs are shown in the SI for the solution conformation of all three peptides.

Once the solution conformation was determined for peptide 2, the design of several additional peptides was proposed to test the robustness of the RDC determined conformer. The synthesis of three peptides which each contained an N-Me at each of the sites that would disrupt one of the IMHBs was quickly completed. As expected, peptides containing a N-Me at dCha4-NH (peptide 3), Phe5(4-F)-NH (peptide 4) and Nle 6-NH (peptide 5) were all inactive in the C5aR1 functional assay measuring TNFα/C5a-driven oxidative burst in human whole blood (Table 1). In addition, changes were made to modify the ornithine chain which we hoped would not disrupt the IMHBs, thus resulting in peptides 6, 7, and 8. A range of activities was observed for these peptides, but all were active as C5aR1 antagonists. As an example of how the C5aR1 homology model and peptide RDC solution conformations can work together, it is interesting to consider the case of peptide 7. This design was originally synthesized and tested as its epimer at the residue 2 (Orn-NδMe) pyrrolidine C4 but found to be only very weakly active (data not shown). When this peptide was examined in the proposed binding site of the homology model, it appeared that the opposite stereocenter on C4 for the pyrrolidine ring gave a better fit. When peptide 7 was synthesized with the stereochemistry as depicted in Table 1 it was found to be more potent than it epimer, as we had predicted, and more potent than peptide 1. In addition, the piperidine ring shown in peptide 8 was also modeled in the homology model and gave a reasonable fit. When this peptide was synthesized it was also found to be active but less potent than peptide 7. This highlights a limitation of this method’s ability to predict relative potencies of analogs.

Comparison of the assigned proton spectra from peptides 2 and 6 (see SI) illustrates that several of the NH resonances have shifted considerably: dCha4-NH, Phe5(4-F)-NH, and Phe6-NH, suggesting that the conformation of peptide 6 is different from peptide 2. Peptide 6 contains a β-phenylalinine residue and the ornithine residue is shortened by one carbon such that the peptide macrocycle is of the same size as peptide 2, but the position of the tertiary amide is adjusted within the macrocycle. Fortunately, peptide 6 had a trans/cis ratio of 20:1 at the tertiary amide, making it an excellent candidate for the next RDC solution conformation determination.

In a similar fashion to the previous determination, the 9 RDCs for peptide 6 were fitted to a family of ∼1,000 low energy conformers using MSpin software.32 The conformer with the best fit had two IMHBs: dCha4-NH to β-Phe6 C=O and β-Phe6-NH to β-Phe6 C=O as shown in Figure 4c. The single conformer with the best fit to all of the RDC data is shown in Figure 4d. In addition, three long-range NOEs were observed for this peptide (dCha4-NH to Pro2-CH2, dCha4-NH to Orn2-CH2, and dCha4-NH to Phe6-NH) which are also consistent with the conformer shown in Figure 4d. Examination of the Tc’s for peptide 6 are consistent with four potential IMHBs: one for each NH in the peptide. From the RDC solution conformation of peptide 6, dCha4-NH and Phe6-NH are believed to be involved in IMHBs and Phe(4-F)5-NH and Me-Phe1-NH from side chain residue 1 are believed to be solvent shielded in the solution conformation and not actually involved in IMHBs. This is a limitation of using Tc’s alone to determine IMHBs. Some exchangeable NHs have a low Tc due to solvent shielding which prevents solvent exchange with temperature in this experiment and may give a false indication of an IMHB for the NH. For this reason, we always combine Tc’s with RDC measurements for confirmation of an IMHB.

Once the solution conformation was determined for peptide 6, several additional peptides were designed and synthesized. Previously, N-methylation at Phe(4-F)5-NH (peptide 4 in Table 2) had yielded an inactive peptide. This was consistent with our predictions of IMHB pattern, as described earlier. Since the β-Phe-containing ornithine for peptide 6 did not exhibit a IMHB using Phe(4-F)5-NH in the solution conformer, yet retained activity at C5aR1, this suggested the potential for N-methylation at Phe(4-F)5-NH may be tolerated. Consistent with our model, the β-alanine-containing peptide 9 retained activity for C5aR1.

Searching for other residues which would also yield a favorable trans/cis ratio for a RDC solution conformation led to the substitution of Pro3 with a threonine residue, peptide 10. The 9 RDCs for peptide 10 were fitted to a family of ∼1,000 low energy conformers using MSpin software. The conformer with the best fit had three IMHBs: Thr3-NH to Phe6 C=O, Phe(4-F)5-NH to Thr3 C=O, and Phe6-NH to The3 C=O (Figure 4e). The conformer with the best fit to all of the RDC data is shown in Figure 4f. In addition, two long-range NOEs were observed for this peptide (Thr3-CH to Phe6-CH2 and Thr3-CH3 to Phe6-CH2), which are also consistent with the conformer shown in Figure 4f. Examination of the Tc’s for peptide 3 are consistent with three potential IMHBs: Thr3-NH, Phe(4-F)5-NH, and Phe6-NH. For peptide 10, dCha4-NH and Orn2-NH also exhibit low Tc’s but are believed to be solvent shielded and not involved in IMHBs.

Once the solution conformation was determined for peptide 10, several additional peptides were designed and synthesized. As expected, peptides resulting from the methylation of NHs that we predicted would disrupt an IMHBs as compared to peptide 10 were found to be inactive (data not shown). Peptide 11 is a combination of the β-alanine-containing peptide 6 and the threonine residue from peptide 10 with a different acyl substituent at residue 1. Combining these two changes yielded the active peptide 11. While the potency of peptide 11 is not greater than that of the initial peptide 1, combining several residue modifications simultaneously demonstrates that multiple modifications that can be made while still maintaining potency and membrane permeability.

This method of using both RDCs and Tc’s to determine solution conformations and guide compound design is now used routinely and has impacted many projects at Pfizer.25,27,33,34 In addition, using solution conformations in combination with a computational model to guide design and synthesis minimizes investment in inactive peptides and has the additional advantage of mapping out the SAR for future design and synthesis.

Acknowledgments

The study was funded by Pfizer, Inc. The authors thank Mark Noe, Arvinda Munasinghe, and Adam Gilbert for many helpful conversations during the preparation of this manuscript and the Chinese Peptide Company for peptide synthesis support.

Glossary

Abbreviations

RA

rheumatoid arthritis

RDC

residual dipolar coupling

NOE

nuclear Overhauser effect

C5a

74-residue anaphylatoxin peptide

IMHB

intramolecular hydrogen bond

SAR

structure–activity relationship

JSB

J-scaled Bird

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00316.

  • Solid phase synthesis of linear and cyclic peptides; general analytical information: HRMS, NMR line lists, 1H NMR, 13C NMR, HC-HSQC, and HN-HSQC spectra of peptides 2, 6, and 10, and proton spectra of remaining peptides; MSpin input files for RDC data collection as well as computation files (PDF)

  • Peptide 2 in chloroform (PDB)

  • Peptide 6 in chloroform (PDB)

  • Peptide 10 in chloroform (PDB)

The authors declare no competing financial interest.

Supplementary Material

ml4c00316_si_001.pdf (1.6MB, pdf)
ml4c00316_si_002.pdb (18.9MB, pdb)
ml4c00316_si_003.pdb (17.6MB, pdb)
ml4c00316_si_004.pdb (71.9MB, pdb)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml4c00316_si_001.pdf (1.6MB, pdf)
ml4c00316_si_002.pdb (18.9MB, pdb)
ml4c00316_si_003.pdb (17.6MB, pdb)
ml4c00316_si_004.pdb (71.9MB, pdb)

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