Jordan M. Mattheisen,Chris Limberakis,Roger B. Ruggeri,Matthew S. Dowling,Christopher W. am Ende,Emilie Ceraudo,Thomas Huber,Christopher L. McClendon,Thomas P. Sakmar
Bioorthogonal Tethering Enhances Drug Fragment Affinity for G Protein-Coupled Receptors in Live Cells
G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C–C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels–Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.
Bioorthogonal Tethering Enhances Drug Fragment Affinity for G Protein-Coupled Receptors in Live Cells

G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C–C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels–Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.

The superfamily of heptahelical G protein-coupled receptors (GPCRs) is a target for more than one-third of FDA-approved drugs. Most small molecules that target GPCRs bind in the orthosteric ligand-binding site to antagonize the effects of endogenous agonists. In the past decade, structural biology studies of GPCRs have revolutionized drug discovery strategies in cases where well-defined drug-binding pockets can be identified in high-resolution structures. However, many allosteric drug binding sites, which are spatially distinct from endogenous ligand-binding sites, may be “cryptic” and accessible only under specific physiological conditions. Molecular dynamics (MD) studies predict the existence of dynamic state-dependent allosteric binding sites in some GPCRs, which provide novel pockets for modulating GPCR pharmacology and opportunities for drug discovery efforts. Allosteric modulators are desirable since they can be more selective even among closely related receptors and their effects can be tuned in the presence of endogenous agonists. Despite the potential benefits of allosteric GPCR modulators, they remain challenging to discover and optimize. Fragment-based drug discovery (FBDD) is a useful method for probing putative drug binding sites and discovering therapeutic lead compounds. Typically, FBDD involves screening a small library of low molecular-weight compounds against a purified protein target using in vitro biophysical assays. GPCRs remain challenging targets for biophysical screens in FBDD due to their relative instability when removed from a native membrane environment. Most GPCR FBDD campaigns reported to date have relied on biophysical assays employing mutagenized receptors optimized through rounds of screening and selection to enhance stability while retaining the desired pharmacological properties. GPCR stabilization still remains a challenge due to the lack of generic and effective stabilization methods. Therefore, functional screens of GPCRs in cell-based signaling assays remain an important element of the drug discovery pathway. One of the first examples of live-cell FBDD for a GPCR target employed a high-content screen of human adenosine-A1 and -A3 receptors using fluorescent ligands and competition with high concentration (1 mM) fragments. Virtual screening in conjunction with cell-based high concentration screening of fragments has also been carried out to identify agonists for the adrenergic α2C receptor. Another study used a drug-fragment library biased toward larger fragments and carried out a cell-based functional screen of GPR7 (neuropeptides B/W receptor type 1). But overall, high-concentration screening in cells is limited by the solubility of screening compounds and often results in high-false positive hit rates due to off-target effects. Covalent tethering of fragments or drugs to targets can be used to enhance their affinity and enable drug screening at lower concentrations. Traditional tethering approaches require native or engineered cysteines in the target protein which undergo thiol–disulfide exchange forming reversible covalent bonds with disulfide-containing screen compounds, which are subsequently identified using mass spectrometry, a task still not trivial for membrane proteins. Cysteine tethering has been employed for screening peptide ligand analogues against the complement fragment 5 anaphylatoxin (C5a) receptor in competition binding assays performed in membranes. Nonetheless, cysteine residues are relatively abundant in GPCRs and are often critical sites for disulfide bond formation and post-translational modifications. Therefore, for site-specific cysteine targeting, cysteine-free receptors must be engineered and functionally validated prior to screening. In addition, the kinetics of sulfhydryl labeling is slow, which means that the cysteine target needs to be close to the drug binding site. An alternative strategy for covalent tethering with minimal receptor perturbation is to use genetic code expansion (GCE) whereby reactive noncanonical amino acids (ncAAs) are site-specifically introduced into protein targets expressed in live cells by engineered orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pairs. Cells expressing the protein target harboring the ncAA can be screened against heterobifunctional compounds, each containing a reactive functional group linked to a small molecule ligand or drug fragment. In theory, bioorthogonal reaction between the ncAA and heterobifunctional compound should result in irreversible covalent tethering of the compound to the target, increasing the effective local concentration and affinity of the compound and enabling detection of target engagement in live cell-based pharmacological assays. This strategy has been used for covalent tethering of MEK inhibitors to regulate protein function in live mammalian cells. Here we present a proof-of-concept study employing GCE and bioorthogonal chemistry in live cells to tether low affinity drug fragments to GPCRs. We hypothesized that bioorthogonal tethering to genetically encoded ncAAs would drive target engagement and therefore target specificity of fragment-like compounds. We demonstrate covalent tethering of heterobifunctional ligands containing one of four reactive functional groups linked to fragments of the allosteric antagonist maraviroc (mvc) to the GPCR C–C chemokine receptor 5 (CCR5) in live cells. In the same cells, we carried out signaling assays to assess target engagement of high- and low-affinity mvc analogues at the engineered CCR5. Using MD simulations, we correlated efficiency and coupling of analogues with the location of ncAA in CCR5. Our study demonstrates the robustness of GCE and bioorthogonal coupling reactions to covalently link ligand analogues and low-affinity drug fragments to engineered GPCRs expressed in culture. The strategy we describe should be useful as a tool to modulate GPCR function using tethering of known ligands, as well as to enable FBDD to target novel allosteric and “cryptic” drug-binding sites in cell-based assays.

Our aim was to develop a general strategy to tether drug fragments to GPCRs to facilitate the study of allosteric or cryptic drug binding sites and to enhance the utility of fragment-based drug discovery using cell-based assays. As a proof-of-concept for our approach, we used GCE to incorporate the ncAA cyclopropene-l-lysine (CpK) site-specifically into a GPCR. The CpK was then used as a chemical handle to react with a heterobifunctional ligand analogue (Figure 1). Cpk was chosen because it would be amenable to the bioorthogonal inverse electron demand Diels–Alder (IEDDA) coupling reaction (Figure 2A). The IEDDA reaction displays relatively rapid kinetics and is compatible with mammalian cells in culture. We selected CCR5 for study because it is a well-characterized GPCR target with a defined binding pocket for the allosteric antagonist drug mvc, and because of our earlier collective experience in structure–function studies and medicinal chemistry on CCR5. We introduced the amber (amb) codon into CCR5 at codon position 184 using site-directed mutagenesis. The 184 position was chosen because it lies in the extracellular loop 2 (ECL2) of CCR5 just above the binding pocket for mvc and mutation of this position was not expected to alter receptor activity (Figure 2B). We used GCE with the Methanosarcina bakeri pyrrolysyl-tRNA synthetase (PylRS)-tRNA pair to suppress the amber codon. We cotransfected the PylRS-tRNA construct on a single plasmid with the CCR5-Y184amb mutant in HEK293T cells in the presence or absence of CpK (Figure 2C). We then carried out agonist dependent Ca2+ mobilization assays to show that the amber codon suppression resulted in functional CCR5 only when the PylRS-tRNA was expressed in the presence of CpK (Figure 2C). Introduction of CpK at position 184 caused only a minor right-shift of the EC50 curve for the agonist RANTES, or the superagonist PSC-RANTES (Figure S1). In addition, CpK did not significantly affect the ability of mvc to inhibit agonist-dependent Ca2+ mobilization. The extent of Ca2+ mobilization provides a measure of the overall efficiency of using the PylRS-tRNA pair to create functional CCR5-Y184CpK. The yield of functional CCR5-Y184CpK was approximately 80%.

Next, we wanted to demonstrate the ability to covalently tether ligands to a GPCR engineered to contain an ncAA in live-cells. Using the known CCR5 allosteric antagonist drug mvc, we designed and synthesized six heterobifunctional mvc analogue ligands designated as M1 through M6. Each analogue included an mvc pharmacophore moiety at one end, a variable-length polyethylene glycol (PEG) linker in place of the difluorocyclohexanyl group of mvc, and a reactive tetrazine or methyltetrazine moiety at the other end (Figure 3A, Figure S2). Tetrazine or methyltetrazine was chosen as the reactive moiety in the compounds to facilitate the IEDDA coupling reaction with CpK introduced into the CCR5-Y184CpK variant (Figure 2A). We also synthesized a series of heterobifunctional compounds in which either the dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]non-4-yne (BCN) moiety replaced tetrazine or methyltetrazine (Figures S3 and S4). These compounds were designed to react with the ncAA 4-azido-phenylalanine (azF) using the strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC) reaction. Although azF could be readily introduced into CCR5 using GCE with an engineered tyrosyl-tRNA synthetase (TyrRS)-tRNA pair, we found that the IEDDA reaction was superior to the SPAAC reaction due to relatively rapid kinetics and better compatibility with mammalian cells in culture. In addition, unlike the IEDDA reaction, SPAAC between GPCRs and BCN- or DBCO-containing compounds has been shown to result in cross reactivity with thiols and slow reaction kinetics particularly at solvent-exposed residues. Therefore, we focused on the PylRS system and the IEDDA coupling reaction. A direct comparison of the two chemistries is presented elsewhere. We carried out molecular dynamics (MD) docking simulations using models of M5 and M6 covalently bound to CCR5-Y184CpK, which were built up from the crystal structure of the CCR5-mvc complex (PDB: 4MBS). We analyzed a set of six replicate simulations of each complex by clustering the protein–ligand conformations based on the coordinates of the CpK residue and mvc analogue using GROMACS’ “gromos” method and a 2-Å cutoff and then aligning the cluster representative snapshots using PyMOL (Figure 3B). For both compounds, we found that the bound mvc pose in the CCR5 ligand-binding pocket was largely maintained, while more varied PEG positions were observed with the longer PEG linker in M6 compared with M5. This observation was also supported by the greater relative per-atom root-mean-square fluctuations (RMSF) in the M6 linker as expected due to its greater number of PEG units (Figure S5). We expected that long linkers would be advantageous for FBDD since they would allow tethering to anchor points distant from the targeted protein binding site, thereby minimizing perturbations to that site.

We next assayed the pharmacological activity of the M1 through M6 heterobifunctional compounds in Ca2+ mobilization assays to measure their ability to inhibit superagonist (PSC-RANTES)-induced Ca2+ mobilization in cells expressing either CCR5 or CCR5-Y184CpK (Figure 3C). In control experiments, mvc inhibited PSC-RANTES-induced Ca2+ mobilization in a dose-dependent manner with IC50 values less than 10 nM for both CCR5 and CCR5-Y184CpK. The mvc analogues M1 through M6 also inhibited Ca2+ mobilization in cells expressing either CCR5 or CCR5-Y184CpK. As expected, in cells expressing CCR5, the IC50 dose–response curves for all the mvc analogues were right-shifted compared with the dose–response curve for mvc. In contrast, in cells expressing CCR5-Y184CpK, compounds M2, M4, M5, and M6 displayed left-shifted dose–response curves, indicating higher apparent affinities for the CCR5-Y184CpK variant compared with CCR5. To estimate the contribution of tethered and untethered analogues to the observed antagonist activities and to remove experimental variability in transfection efficiency and compound potency due to ligand concentration and incubation time, we determined a nominal “beta” value for each compound. The beta value effectively estimates the ability of the tetherable antagonist to have enhancement in antagonist activity specifically when able to tether (i.e., when the noncanonical amino acid is present with Y184CpK) (Figure 3C; see also Figure S4). Beta is determined by subtracting the effect of introducing the ncAA on the ability of mvc to bind (ΔpIC50 (amb–wt), mvc)) from the effect of the tetherable mvc analogue to exert a greater effect on the ncAA-containing receptor compared with the wt receptor (ΔpIC50 (amb–wt), compound)). Four of the compounds (M2, M4, M5, and M6) had significant positive beta values, suggesting an enhancement in potency from the combination of Y184CpK with the ligand’s phenyl tetrazine warhead. For example, the ability of M6 to inhibit Ca2+ mobilization in cells expressing CCR5-Y184CpK was approximately 10-fold greater than in cells expressing CCR5. Interestingly, some compounds, especially M2 and M5, which have medium-length linkers, were not able to fully suppress PSC-RANTES-induced Ca2+ mobilization even at high apparent concentrations. This apparently incomplete antagonism is a characteristic of allosteric, noncompetitive antagonists. We found that the ability of certain heterobifunctional compounds to inhibit agonist-induced Ca2+ mobilization was enhanced in CCR5-Y184CpK as predicted by the MD simulations that indicated stability of the covalently bound mvc pose. In compounds M1–M6, the mvc moiety was the same in all cases, but the linker length and reactive group varied. The results above suggested that covalent tethering was enhancing the apparent affinity of the compounds. A linker length of four PEG units was optimal, and methyltetrazine was a better reactive group than tetrazine in our assay system (Figure 3). We hypothesize that although the reaction kinetics of methyltetrazine is expected to be slower, its serum and cellular stability are greater. We did not investigate this observation further.

The data from M1–M6 support the hypothesis that covalent tethering of a mvc analogue to CCR5-Y184CpK should increase its potency for inhibition compared with CCR5 where covalent tethering is not possible. To test this hypothesis further, we designed and synthesized six additional heterobifunctional mvc analogues that were expected to have lower affinities for CCR5 (M7–M12, Figure S2). Due to the greater potency and greater flexibility of the four PEG-unit linkers over the two PEG-unit linkers, we focused on using the longer linkers and trimmed back one or more parts of the mvc to weaken noncovalent association with CCR5. In addition, since the methyltetrazine-containing compounds from the first cell-based screen had slightly higher beta values compared with tetrazine-containing compounds (Figure 3), we chose to include methyltetrazine tethering moieties in each of the lower-affinity mvc analogues. We then tested compounds M7 through M12 for their ability to prevent RANTES-induced Ca2+ mobilization in cells expressing CCR5 or CCR5-Y184CpK. None of these compounds inhibited Ca2+ mobilization in cells expressing CCR5 at up to 10 μM concentration, but four compounds (M9−M12) partially inhibited Ca2+ mobilization for cells expressing CCR5-Y184CpK (Figure 4). This result suggested that the low affinity mvc analogues could achieve a pharmacological effect only when covalent tethering was possible, and that site-directed tethering of fragments using the present approach was sufficient to achieve measurable pharmacological effects in cells, opening possibilities for broader application of this work in drug discovery projects with cell-based assays.

To investigate the relationship between compound affinity and covalent tethering contributing to pharmacological activity, we devised a fluorescent pulse-chase labeling experiment (Figure 5A). HEK293T cells expressing CCR5-Y184CpK were incubated with mvc, heterobifunctional mvc analogues, or control compounds lacking mvc moieties, but containing methyltetrazine linked to a carboxylic acid group (M13) or an amine (M14) (pulse), prior to incubation with AZDye 680R Tetrazine (Tet-680) (chase) (Figure 5B). Cells were then lysed, and CCR5 was enriched using a C-terminal 1D4 epitope tag ensuring capture of full-length receptor. We then performed immunoblotting against the 1D4 tag of CCR5-Y184CpK and visualized total CCR5-Y184CpK (green) and Tet-680-labeled CCR5-Y184CpK (red) (Figure 5C). Cells expressing CCR5-Y184CpK could be robustly labeled with Tet-680 in the presence or absence of mvc, which shows directly that the IEDDA reaction was satisfactory for bioorthogonal labeling of a GPCR harboring CpK. The presence or absence of mvc did not seem to affect the efficiency of the labeling reaction, suggesting that the mvc did not inhibit the reaction. These results confirmed that residue 184 at the receptor surface is a sufficient distance from the mvc binding pocket and suggests that mvc is not required to induce a particular CCR5 conformation amenable to reaction. Pretreatment with two of the low-affinity mvc analogues (M7 and M8) did not significantly block Tet-680 labeling. This result is consistent with the inability of M7 and M8 to inhibit RANTES-induced Ca2+ mobilization in functional screens of cells expressing CCR5-Y184CpK (Figure 4). Pretreatment with the four other low-affinity mvc fragment compounds (M9–M12) significantly prevented Tet-680 labeling. These compounds, unlike M7 and M8, were pharmacologically active in Ca2+ mobilization assays using CCR5-Y184CpK, but not when using CCR5 (Figure 4). The high-affinity tetherable mvc analogues M5 and M6, which inhibited agonist-induced Ca2+ mobilization in cells expressing CCR5 or CCR5-Y184CpK, also blocked Tet-680 labeling. Pretreatment with M13 and M14 compounds, which can tether but lack mvc affinity, did not prevent subsequent Tet-680 labeling, implying that a requirement for a mvc fragment, even one with low-affinity, is needed to enhance reactivity with CCR5-Y184CpK. Together, these data confirm that the pharmacological findings for CCR5-Y184CpK compared with CCR5 are due to covalent tethering of the otherwise low-affinity mvc fragments. The data also suggest that the affinity of the drug fragment plays a critical role in the ability of the heterobifunctional compound to form a covalent complex with the target protein, which is advantageous for fragment-based screening as discussed below.

As is the case with any covalent heterobifunctional compound, there is an interplay between the ligand affinity and the tethering reaction rate. To understand these contributions on our system and further validate our experimental findings, we carried out mathematical modeling of receptor occupancy as a function of ligand concentration and time (Figure S6). The Kd of a drug fragment of between 200 and 300 Da would typically be on the order of 1 mM, and the effects of these low-affinity fragments would generally not be able to be measured in cell-based assays. Our modeling showed that with compound incubation times typical for cell-based assays, on the order of a few hours, there was no apparent difference in the dose-occupancy curves for high-affinity covalent or noncovalent ligands with slow-on rates. This observation is consistent with the relatively small beta values that we calculated in our initial high-affinity mvc analogue screen. Our modeling also indicated that the tethering reaction kinetics have a large impact on the receptor occupancy for low-affinity ligands. For example, for systems using fast reactions (i.e., trans-cyclooct-2-en-l-lysine (TCOK) and tetrazines) the tethering reaction can proceed in the solution phase without need for transient ligand binding. In contrast, our system employs a relative slow IEDDA reaction between CpK and tetrazines, necessitating transient ligand binding to enable proximity ligation resulting in subsequently high local concentration of the ligand and a measurable pharmacological effect in cell-based assays. In principle, this observed ligand-affinity-driven tethering is advantageous to select for fragment hits that have specific affinity for the binding site.

We next asked whether a computational approach could help predict which sites on the protein might be most suitable for introduction of CpK to allow satisfactory linkage of mvc analogues. Due to the large number of rotatable bonds in the CpK, PEG linker, mvc fragment, and nearby protein backbone, a standard covalent docking and prediction method to obtain a pose for the CpK-conjugated mvc analogues was not feasible. Instead, we took advantage of the fact that the noncovalent mvc fragment pose was quite stable, while the CpK and even the nearby CCR5 backbone could be quite flexible. We selected 14 additional candidate residues on the extracellular surface of CCR5, including positions facing away from the mvc binding site (F96, H88, and G265) and one putative negative control residue on the intracellular surface (L218). We performed a 500 ns MD simulation for each of the 15 CCR5-CpK variants in the presence of bound mvc, visualized the end point of each simulation to contextualize the observed distance distributions (Figure 6A), and recorded the distribution of distances between the proximal carbon on the cyclopropenyl ring of the CpK and the amide carbonyl carbon of mvc observed in the simulations (Figure 6B). We then created the 15 CCR5-CpK variants experimentally and measured RANTES-induced Ca2+ mobilization in live cells for each CCR5-CpK variant after incubation with low or high concentrations of mvc or tetherable fragment M12 (Figure 6C). All of the receptor variants were functional as judged by agonist-induced Ca2+ mobilization. Pretreatment with mvc totally inhibited Ca2+ mobilization in every mutant except Y108CpK. Analogue M12 did not block RANTES-induced Ca2+ mobilization in CCR5 (Figure 4). For the CCR5 variants, the ability of M12 to block Ca2+ mobilization depended on the location of the CpK. In general, if CpK was either too close or too far from the mvc binding pocket, M12 had no effect on Ca2+ flux. For example, mutants F109CpK, Y108CpK, and W86CpK, in which CpK was very close to the mvc binding site, or L218CpK, in which CpK was far from the mvc binding site, behaved similarly to the nonmutated CCR5. M12 was not able to block Ca2+ mobilization in these mutants. The exception in terms of CpK-mvc distance was G265CpK, where the CpK side chain in the model points predominantly away from the mvc binding pocket. We then measured the degree of covalent reaction of M12 with each of the 15 CpK receptor variants using the pulse-chase assay. Quantitation for the gel insets in Figure 6D is presented in Figure S7. In general, CCR5-CpK mutants that were responsive to M12 treatment in the Ca2+ mobilization assay also showed reactivity with M12, thereby blocking Tet-680 labeling (Figure 6D). Although the G265CpK, H88CpK, and D95CpK mutants did not show inhibition of agonist-induced Ca2+ mobilization upon pretreatment with M12, they all showed significant reactivity as indicated by high levels of Tet-680 labeling with and without M12 treatment. In general, the dynamic distance distributions from the ncAA to the mvc binding site confirmed the expected distance and steric limitations for the M12 compound to form covalent complexes with the ncAA. CpK mutation positions that yielded significant pharmacological activity tended to have substantial density in the intermolecular distance range of 14–20 Å, consistent with the intramolecular distance distribution for the same atoms of the CpK-M6 conjugate in the simulations. Closer approaches were also possible provided that there would be room for the linker and tethering functional group, for example with F109CpK and Y184CpK. Most variants with distance ranges greater than 20 Å did not show functional response to M12. One exception was Q93CpK, which was functionally responsive to M12 despite having most distances in MD simulations larger 20 Å, likely due to a limited sampling of distances shorter than 20 Å.

We developed a strategy using GCE and bioorthogonal chemistry to tether ligands to expressed GPCRs in live cells. Using pharmacological second-messenger assays amenable to high-throughput drug screening, we were able to directly monitor the effect of the tethered ligands without perturbation of the native GPCR cellular environment required for typical biophysical assays. The GCE approach achieves single-residue selectivity for compound tethering without the need for cysteine-free mutagenesis or thermostabilization of the receptor. By using rapid covalent bioorthogonal tethering reactions, we were able to use longer linkers in the heterobifunctional compounds than would be possible with cysteine labeling approaches, enabling placement of the tethering site relatively far away from the ligand pocket so as to not affect fragment binding. The system described allows expression of the receptor, bioorthogonal tethering of drug fragments, and assay of pharmacological effects all to be carried out on cells from a single well of a microtiter plate. We believe that our system can potentially select for transient ligand-binders through a ligand affinity-enhanced IEDDA covalent tethering reaction, which enhances ligand local concentration resulting in a measurable pharmacological effect in live-cell assays. Although we demonstrated pharmacological activity using Ca2+ mobilization assays, it is possible for alternative pharmacological assays to be used. One potential limitation of the assay format is that ncAA present in cellular media during assaying acts as a scavenger of the reactive reagents thereby reducing the fraction of receptors tethered to the heterbifunctional compounds. We tested a limited number of tethering sites mostly on the extracellular loops of the receptor. The use of transmembrane or intracellular sites for heterobifunctional compound tethering would require additional assessment of appropriate bioorthogonal reactions and analogue linkers to ensure cell-permeability and accessibility to the receptor sites. Success of the method relies directly on the efficient incoporation of ncAA that is stable when introduced into the protein, but is sufficiently reactive to undergo bioorthogonal coupling reactions. GCE technology and bioorthogonal chemistry continue to be improved for use in cellular and in vivo systems. While we used CCR5 and drug fragments based on the known structure for its allosteric antagonist mvc to demonstrate proof-of-principle for the approach, we believe our strategy will be useful to probe the targetability of state-dependent and dynamic GPCR cryptic sites, which have been computationally predicted to exist, but whose functional or drug-target relevance is not known. Tethering novel ligands or drug fragments can enable fragment-based drug discovery using live-cell screening and potentially circumvent the need for tedious protein purification and traditional biophysical fragment screening methods. When combined with computational or structure-based approaches, fragment hits can then be elaborated or linked to achieve high-affinity lead compounds with the desired pharmacology.

The plasmid CCR5–1D4 contains the human coding sequence of CCR5 and the C-terminal 1D4 epitope tag (TETSQVAPA) in a pcDNA3.1(+). QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) was used to engineer amber codon (amb, UAG) mutations at positions K22, I23, W86, H88, Q93, D95, F96, Y108, F109, F182, Y184, Q188, L218, E262, G265, N267 in CCR5-1D4. Oligonucleotides for mutagenesis were purchased at the standard desalting grade (Integrated DNA Technology). Coding sequences were confirmed using sanger sequencing. Amber codon suppression machinery (aaRS/tRNA pairs) for incorporating azF consisted of one plasmid encoding the Bst-Yam amber suppressor tRNA derived from Geobacillus stearothermophilus (tRNATyr) and one plasmid encoding the evolved Escherichia coli TyrRS(Y37L/D182S/F182M/L186A) as described previously. For CpK incorporation, a single plasmid containing four copies of U6 promoter-driven suppressor tRNAs and one copy of a CMV promoter-driven Methanosarcina bakeri PylRS provided by Simon Elsässer was used. Gqi5 refers to a plasmid encoding a modified α subunit of Gq where the C-terminal tail amino acid residues are derived from the α subunit of Gi2 (such that EYNLV-COOH in Gq becomes DCGLF-COOH in Gqi5) engineered in pcDNA3.1(+).

HEK293T cells (American Type Culture Collection, CRL-3216) were cultured in Dulbecco’s Modified Eagle Medium with 4.5 g/L d-Glucose (DMEM-GlutaMax) (Thermo Fisher Scientific) supplemented with 15 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5 and 10% (v/v) BenchMark fetal bovine serum (FBS) (Gemini). Cells were maintained at 37 °C and 5% CO2 with cell passage number <20. Transfections were performed at 70–80% confluency using 2.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) per μg of plasmid DNA. Total DNA was kept constant using empty vector pcDNA3.1(+) when necessary.

4-Azido-l-phenylalanine (azF) (Chem-Impex International, Inc.) stocks were prepared to 0.5 M concentration in 0.5 M NaOH and stored at −20 °C until use. N6-[[(2-Methyl-2-cyclopropene-1-yl)methoxy]carbonyl]-l-lysine (CpK) (Sirius Fine Chemicals) stocks were prepared to 100 mM in 0.2 M NaOH/15% dimethyl sulfoxide (DMSO) solution and stored in aliquots at −20 °C. CpK stocks were diluted to 20 mM in 1 M HEPES just before use. Cell culture media containing azF and CpK was filter-sterilized prior to addition to cells at a final concentration of 0.5 mM.

12 h prior to transfection 2 mL of DMEM containing ∼700,000 cells were seeded into 6-well plates. Cells were transfected with 3 μg of total plasmid DNA. For CpK incorporation, ratios of receptor:PylRS/tRNA:Gqi5:pcDNA DNAs were 1:1:0.5:0.5. For azF incorporation, the ratios of receptor:TyrRS:tRNA:Gqi5 DNAs were 1:0.5:1:0.5. Briefly, DNA and Lipofectamine 2000 were diluted separately in DMEM (no FBS) and incubated at rt for 5 min. Then DNA and Lipofectamine 2000 were combined in equal volumes and incubated at rt for 20 min prior to dropwise addition to the preseeded cells. After 4–6 h, 1 mL of media was removed from each well and replaced with 1 mL of DMEM supplemented with 1 mM [2X] prepared ncAA. After 24 h cells were dissociated from the plates using trypsin-EDTA (0.25%, phenol red, Thermo Fisher Scientific) and resuspended in DMEM supplemented with 0.5 mM ncAA. Cells (∼20,000) were seeded in 20 μL of DMEM supplemented with 0.5 mM ncAA into a 384-well microtiter plate (polystyrene, black-walled, flat bottom, Greiner Bio-One) previously coated for 1 h with 0.01% (w/v) poly-d-lysine (PDL) hydrobromide (Sigma-Aldrich) and washed. The plate was returned to 37 °C, 5% CO2 overnight.

FLIPR Calcium 6 dye (Molecular Devices) was reconstituted to [4X] concentration in HBSS-H (HBSS + 20 mM HEPES) with 0.4% (w/v) bovine serum albumin fraction V fatty acid-free (BSA). Then 10 μL of the dye were added to each well of the assay plate and incubated for 2 h at 37 °C, 5% CO2. For the agonist dose–response assays, 10 μL of assay buffer (HBSS-H + 0.4% BSA) were also added. For the mvc and mvc analogue competition assays, serial dilutions of mvc or mvc analogues were prepared in DMSO prior to dilution in assay buffer at [5X] concentration and addition of 10 μL to the cells (10 pM to 10 μM final). The mvc compounds were incubated with the cells and calcium dye for 2 h at 37 °C. The assay plate was transferred to a prewarmed (37 °C) FlexStation II 384 Plate Reader (Molecular Devices) 30 min prior to injection of 10 μL of [5X] agonist (RANTES, regulated on activation, normal T-cell expressed and secreted chemokine; PeproTech, Inc. or PSC-RANTES, (Nα-(n-nonanoyl)-des-Ser1-[l-thioprolyl,l-cyclohexylglycyl]-RANTES(4-68)). The final concentration of agonist for the mvc competition assays was 0.1 μM. Fluorescence readings were collected by the instrument with excitation 485 nm, emission 535 nm, and the dichroic mirror at 525 nm over a 100 s time course, with agonist added to the cells with mixing 20 s after the start of measurement. Relative fluorescence units (RFU) were calculated for each well as either (1) the mean signal between 20 and 100 s (raw injection signal) minus the mean signal between 0 and 20 s (basal signal) or (2) the max–min signal for the whole read. Values were plotted in GraphPad Prism 9.1.0. The best-fit model was selected between a horizontal line versus a three-parameter fit for each of the dose–response curves using Akaike’s Information Criterion. Fit parameters can be found in Table S1 of the Supporting Information.

For each mvc analogue screened, we calculated a nominal “beta” value to quantify the relative potency of inhibition of agonist-induced Ca2+ mobilization of CCR5 wt versus CCR5-Y184CpK amb using the formula: beta = Δ(ΔpIC50 (amb – wt), compound M#) – Δ(ΔpIC50 (amb – wt), mvc). For visualization, RFU signals (max-min) were normalized to the value 1 relative to the receptor (either CCR5 wt or amb mutant) in the presence of mvc. Normalized values were plotted using a three-parameter fit model with the top parameter constrained to 1 or as a horizontal line with the mean constrained to 1 (see Table S2 for detailed beta value calculations).

Models for MD simulations were prepared using Schrödinger’s Protein Preparation tool in the Maestro program beginning with the crystal structure of CCR5 in complex with mvc (PDB: 4MBS). The protonation states of residues were predicted using propka, and minimization was restrained to at most 0.3 Å RMSD using the OPLS4 force field. The cytosolic insertion (residues 1001–1054 from PDB: 4MBS) was deleted to yield the reference model. Next, CpK plus mvc analogue conjugates were modeled. As these mvc analogue conjugates to CpK have too many rotatable bonds for the covalent docking protocol, they were created as follows: the Y184 position was mutated to CpK, and the mvc portion was fixed, while the initial positions of the CpK side chain and intervening linkers with two PEG subunits (M5) or four PEG subunits (M6) were manually modeled using the 3D builder toolbox, followed by restrained minimization to at most 0.3 Å RMSD. Additional systems were prepared without the covalent linkage, and with the original mvc ligand as before except that the reference model with mvc bound was mutated to lysine at each position using the residue mutation panel in Maestro, and then the lysine side chain was converted into CpK using the 3D builder tool. The resulting side chain was optimized using Schrodinger’s Prime side-chain optimization panel, again followed by restrained minimization to at most 0.3 Å RMSD.

MD simulations were performed in explicit solvent with the Desmond software package, with the OPLS4 force field. The protein was placed in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane simulation box setting the transmembrane atoms to helices, and automatic placement with a buffer of 10 Å of water on each side (SPC water model) was used. Na+ and Cl– ions were added to neutralize the system, and then 0.05 M NaCl was added. Equilibration was performed using the default protocol for systems with membrane. After equilibration, multiple (different random seed) production runs of 500 ns were performed on each system using the Martyna–Tobias–Klein integrator at 300 K (Nose–Hoover thermostat) with a relaxation time of 2 ps and a pressure of 1 atm, with constrained surface tension and pressure. Snapshots were output every 1.0 ps. All bonds involving hydrogens were constrained, a 1 fs time step for the bonded and short-range nonbonded interactions was used, and long-range nonbonded interactions were updated every 6 fs using the RESPA multiple time step approach, except in the set of simulations with CCR5, noncovalently bound mvc, and varying the mutant CpK residue position, in which case a 1 fs time step and 6 fs RESPA update were used. Short-range Coulombic and van der Waals nonbonded interactions were cut off at 9.0 Å, and long-range electrostatics were computed using the smooth particle-mesh Ewald method.

Cells (∼4 × 106) were seeded in 10 mL of DMEM culture media into 10 cm plates the day prior to transfection. Cells were transfected with 5.6 μg of receptor DNA and 11.2 μg of amber machinery using Lipofectamine 2000 as described previously. After 48 h, media was aspirated from the plates and cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Thermo Fisher Scientific). Cells were incubated with 10 μM pulse compound (DMSO, mvc, M5-M12, or M13-M14 (Click Chemistry Tools)) in FluoroBrite-DMEM (Thermo Fisher Scientific) + 15 mM HEPES, for 2 h at 37 °C, 5% CO2. After incubation cells were scraped from the plates, pelleted (150 RCF, 5 min, 4 °C), washed in 10 mL of cold DPBS, resuspended in 2 mL of labeling medium (FluoroBrite-DMEM, 15 mM HEPES, 50 μM AZDye 680R Tetrazine (Tet-680, Click Chemistry Tools)), and incubated while nutating for 6 h, 4 °C. Then cells were washed in 10 mL of cold DPBS, resuspended in 1 mL of lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1 mM CaCl2, 1% n-dodecyl-β-d-maltopyranoside (DDM), 0.1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich), 0.12 mg (0.552 units) of aprotinin (Sigma-Aldrich), cOmplete Mini, EDTA-free protease inhibitor cocktail (Roche)), and nutated for 1 h, 4 °C. Samples were centrifuged at 21,000 RCF, 30 min, 4 °C to pellet cell debris.

1D4 mAb was coupled to sepharose 4B resin as previously described. The 1D4-sepharose resin was washed several times in DPBS prior to use. Resin slurry (∼50 μL) was added to each Ultrafree-MC-HV Durapore PVDF 0.45 μm centrifugal filters (Millipore) to which the cleared lysates were added and incubated while nutating overnight, 4 °C. The resin was washed 3 times with 30 min incubations in Buffer III + G (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.02% Cholesteryl hemisuccinate (CHS), 0.1% DDM, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate] (CHAPS), and 50 nM 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (7:3), 10% glycerol), prior to elution in 50 μL of elution buffer (Buffer III + G + 0.33 mg/mL 1D5 peptide (TETSQVAPA)).

Immunopurified lysates were incubated with NuPAGE LDS Sample Buffer 4X (Thermo Fisher Scientific) and 100 mM 1,4-dithiothreitol (DTT) prior to gel electrophoresis using NuPAGE 4–12% Bis-Tris gel,1.5 mm × 15 well Mini Protein Gel in NuPAGE MES-SDS Running Buffer (Thermo Fisher Scientific) at a constant voltage of ∼115 V. Gel was transferred to Immobilon-FL PVDF membrane (Millipore) for immunoblot analysis. All incubation steps were 1 h at rt unless noted otherwise. The blocking step was carried out using Intercept Blocking Buffer (IBB, LI-COR Biosciences) prior to primary antibody incubation (IBB + 0.1% Tween 20 + 1:2000 dilution anti-1D4 mouse mAb). Membranes were briefly washed 5 times in DPBS + 0.1% Tween-20 prior secondary antibody incubation (IBB + 0.1% Tween 20 + 1:10,000 IRDye 800CW Goat anti-Mouse IgG (LI-COR Biosciences)). Membranes were washed again and then imaged on LI-COR Odyssey Sa or M infrared laser scanning imagers.

A mathematical model was created to simulate the effects of the ligand on-rate and kinetics of the covalent tethering reaction on receptor ligand occupancy over times from 0.06 to 16 h (Figure S6). A script was written using Mathematica. The local concentration of ligand (Clocal) was set at 5 mM based on an experimentally validated model for effective molarity of a tethered ligand, which was dependent on linker length, steric hindrance, and the distance of anchor point to the binding pocket. Ligand binding affinities (Kd) were set at 1 mM to simulate typical affinities for 200–300 Da fragments or 100 nM to simulate a high-affinity GPCR ligand like mvc with a slower-than-diffusion-limited binding rate constant and slow off-rate. Tethering reaction rates (k2) were set at 0.05, 0.5, or 5.0 M–1 s–1, consistent with a range of reaction kinetics reported for SPAAC, IEDDA between cyclopropene and methyltetrazines and IEDDA between transcylooctene and tetrazines. Ligand on-rates (kon) were set at either 103, 104, 105, or 106 M–1 s–1 to simulate rates consistent with cryptic, allosteric, and orthosteric binding.

For RANTES-induced Ca2+ mobilization for CCR5-CpK variants, 280,000 cells were seeded into 12-well plates in 800 μL of DMEM culture medium 24 h prior to transfection with 1.4 μg of total DNA per well (0.4 μg receptor, 0.4 μg receptor, 0.2 μg Gqi5, 0.4 μg pcDNA) and reseeding into 384-well plates as described previously. The day of the assay cells were treated FLIPR Calcium 6 dye and 10 pM mvc, 10 pM M12, 10 μM mvc, 10 μM M12, or vehicle (DMSO) for 2 h prior to automated agonist addition. RFUs were calculated for each well as the mean signal (described above), which was normalized for each receptor by dividing the values by the average signal of the vehicle-treated wells for that receptor. Three technical replicates were averaged within each experiment. Experimental replicates were then combined into a signal data set, where the replicates were multiplied by the average of all of the vehicle-treated signals (3 experimental replicates each with 3 technical replicates) for each corresponding receptor. These relative signals were plotted in a grouped table in GraphPad Prism 9.1.0.

Cells (∼680,000) were seeded into 6-well plates the day prior to transfection with 2.88 μg of total DNA per well (0.95 μg of receptor and 1.93 μg of amber suppression machinery) as described previously. Pulse-chase was carried out as described above where the pulse was either 10 μM M12 or vehicle. Cells were lysed in 150 μL of lysis buffer prior to immunopurification with 50 μL of 1D4-sepharose resin slurry and elution with 35 μL of elution buffer. Immunoblotting was performed as described previously.

Immunoblots were visualized on a Li-Cor Odyssey Sa instrument and analyzed using LI-COR Image Studio Lite (version 5.2.5). Rectangles of the same size were drawn around the protein band corresponding to full-length CCR5. The background was subtracted using the average top/bottom with a border width of 3. The lane normalization factor (LNF) was calculated for each lane by dividing each lane’s 800-channel signal (total CCR5) by the average signal for the CCR5-wt lanes within a single blot. The 700-channel signal (Tet-680-labeled CCR5) was normalized by dividing each lane signal by the LNF. The normalized signal was then divided by the average 700-channel signal from all lanes across the three immunoblots to give the relative labeling signal value for each condition. The relative labeling signal for three separate experiments were graphed in GraphPad Prism 9.1.0. (Figure S7 and Table S3).