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Inhibition of HIV fusion by small molecule agonists through efficacy-engineering of CXCR4 Christian Berg, Viktorija Daugvilaite, Anne Steen, Astrid Sissel Jørgensen, Jon Våbenø, and Mette Marie Rosenkilde ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00061 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Inhibition of HIV fusion by small molecule agonists through efficacyengineering of CXCR4 Christian Berg1, 2, Viktorija Daugvilaite1, Anne Steen1†, Astrid Sissel Jørgensen1, Jon Våbenø3, and Mette Marie Rosenkilde1, * 1

Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark 2 Infectious Diseases Unit, Department of Medicine, Herlev–Gentofte Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark 3 Helgeland Hospital Trust, Prestmarkveien 1, 8800 Sandnessjøen, Norway ABSTRACT: CXC chemokine receptor 4 (CXCR4) is involved in multiple physiological and pathological processes, notably as a co-receptor for human immunodeficiency virus (HIV) cell entry. Its broad expression pattern and vital biological importance make CXCR4 a troublesome drug target, as disruption of the interaction with its endogenous ligand, CXC chemokine ligand 12 (CXCL12), has severe consequences. In fact, only one CXCR4 drug, the bicyclam antagonist and HIV entry inhibitor AMD3100 (Plerixafor/Mozobil), has been approved for clinical use, however only for stem cell mobilization—a consequence of CXCR4 antagonism. Here, we report the engineering of an efficacy switch mutation in CXCR4—F292A7.43 in the middle of transmembrane helix 7—that converted the antagonists AMD3100 and AMD11070 into partial agonists. As agonists on F292A CXCR4, AMD3100 and AMD11070 were less disruptive to CXCR4 signaling while they remained efficient inhibitors of HIV fusion. This demonstrates that small molecule CXCR4 agonists can have a therapeutic potential as HIV entry inhibitors.

Chemokine receptors are a family of 23 distinct seven-transmembrane (7TM) receptors that mediate signals induced by their chemotactic cytokine ligands (chemokines). Chemokines can be divided into four subgroups based on the distance between conserved N-terminal cysteines: CC, CXC, CX3C, and XC. In contrast to most other chemokine receptors, CXC chemokine receptor 4 (CXCR4) is widely expressed in human tissues outside the immune system1 and is a vital component of several biological processes such as immunity, homeostasis, angiogenesis, and neuronal guidance2–4. Knockout of the receptor causes fatal developmental defects4 and knockout of its only endogenous chemokine ligand, CXC chemokine ligand 12 (CXCL12), leads to pre-natal death in mouse models2, which is not seen in knockout models of other chemokines. Furthermore, CXCR4 is implicated in a variety of pathologies including autoimmune disease, cancer, and human immunodeficiency virus (HIV) infection5–7, where it, together with CC chemokine receptor 5 (CCR5)8, acts as a co-receptor for HIV cell entry through interaction with the viral glycoprotein gp120. Typically, CCR5 is exploited in the earlier stages of HIV infection, but during the later stages a tropism shift towards CXCR4 can occur9, which is correlated with a decline in CD4+ T cell count, development of AIDS, and thus increased mortality10. Since the discovery of these two distinct chemokine receptors’ role in HIV entry, they have been the targets of a large number of potential anti-HIV drugs11. This focus has, however, only resulted in the approval of one such drug for HIV treatment—the small molecule CCR5 antagonist Maraviroc12, 13 (Selzentry/Celsentri). Because of its broad expression pattern and notable physiological importance, CXCR4 is an especially difficult target for drug development.

The small molecule CXCR4 antagonist AMD3100—developed by AnorMED—was intended for use as an anti-HIV drug14, but clinical trials revealed that it mobilized hematopoietic stem cells (HSCs) from the bone marrow to the bloodstream15. This effect, caused by blocking the interaction of local CXCL12 with CXCR4, has subsequently proven useful in harvesting HSCs prior to bone marrow transplantation of certain cancer patients, for which AMD3100 (marketed as Plerixafor/Mozobil) was approved in 2008. Furthermore, AMD3100 has shown some promise as a potential treatment for WHIM (Warts, Hypogammaglobulinaemia, Immunodeficiency, and Myelokathexis) syndrome16, a rare genetic disease caused by different heterozygous CXCR4 mutations. These mutations lead to truncation of the Cterminus, which abolishes normal β-arrestin–mediated receptor downregulation upon CXCL12 stimulation, thus prolonging the induced signal. However, for treating most other CXCR4related disorders, the strategy of using antagonistic compounds remains inherently problematic. Using a CCR5 efficacy switch mutation for the small molecules Maraviroc and Aplaviroc, we recently reported that small molecule agonists can be effective inhibitors of CCR5-tropic HIV fusion17. Given the problems related to blocking of the CXCR4 system with antagonists, a similar agonist strategy would be particularly relevant for CXCR4. We therefore wanted to investigate whether inhibition by small molecule agonists could also be applicable in CXCR4-tropic HIV fusion. Until recently18, no small molecule CXCR4 agonists had been described, so we first set out to design an efficacy switch mutation for the bicyclam AMD3100 and three structurally related


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Figure 1. Structures of small molecule compounds, and the CXCR4 binding pocket with the Phe292 aromatic network. (A) Structures of the bicyclam AMD3100, monocyclams AMD3465 and AMD3389, cyclam ring, and the non-cyclam AMD11070. (B) Receptor structure of WT CXCR4 (PDB code 3OE0)19 showing the location of the residues that were mutated in the present study as well as the aromatic network around Phe292/TM7; - interactions are shown as blue dotted lines.

monocyclams, as well as the non-cyclam AMD11070 (Figure 1A) in CXCR4. AMD11070 was a result of pharmacokinetics/pharmacodynamics optimization of AMD3100 20. It showed longer half-life, oral bioavailability, and maintained anti-HIV activity both in vitro and in vivo20, 21, but ultimately did not make it through clinical trials. Based on the crystal structure of CXCR4 in complex with the viral chemokine vMIP-II22, Wescott et al. have recently proposed an activation model that involves 41 key residues, which together form an intramolecular signaling chain23. Through inspection of the binding pocket and this activation network, we identified Phe2927.43 in TM7 (Figure 1B) as a candidate residue for the efficacy switch. In Wescott’s model, Phe2927.43 was identified as a “signal propagation residue”, and it is a central part of an aromatic network that also involves Tyr45 1.39, Trp942.60, Tyr1163.32 (“signal initiation residues”), and Trp2526.48 (another “signal propagation residue”). Mutation of Phe292 to an alanine will disrupt this network, potentially altering the behavior of TM7 upon ligand binding. Such alterations can in turn allow for binding and activation of G proteins, as we have recently shown for CCR524, 25, converting antagonist into agonists. The functionality of the F292A CXCR4 mutant in terms of G protein signaling was first tested in a Ca2+ mobilization assay using the endogenous chemokine agonist CXCL12 (Figure 2A). The F292A mutation did not affect the efficacy of CXCL12 compared to wild type (WT) receptor, thus the mutated receptor was functionally intact. The surface expression of WT and F292A CXCR4 was also at a similar level (Figure 2B) as evaluated by enzyme-linked immunosorbent assay (ELISA) using a specific antibody towards an N-terminal tag. Next, in order to identify potential agonistic activity of the five small molecules (Figure 1A) on F292A CXCR4, these were all tested for their ability to stimulate and inhibit Gαi signaling. Antagonism of CXCL12-induced activity on F292A was preserved for the three monocyclams (cyclam ring, AMD3465 26, and AMD338927), but none of them were able to stimulate Gαi signaling when added alone (Figure S1). However, the bicyclam AMD3100 and the non-cyclam AMD1107020 both exerted agonistic activity with efficacies of ~50–60% compared to CXCL12 (Figure 2C and D). Furthermore, both compounds inhibited the CXCL12-induced signal to the level of their own intrinsic activity and displayed antagonistic potency in the same range as the agonistic potency (EC50 values of 94 nM vs 139 nM for AMD3100, and 12 nM vs 15 nM for AMD11070, Figure

2C and D). The F292A mutation did not produce any constitutive activity (data not shown). Thus, AMD3100 and AMD11070 both function as partial agonists on F292A CXCR4. In line with the above findings, AMD3100 and AMD11070 were both able to induce Ca2+ mobilization on F292A CXCR4 but not on WT (Figure 2E and F). The two compounds showed similar efficacies, but the signal intensities were weaker than for CXCL12 (Figure 2A), which again indicates partial agonism. The signal in response to AMD3100 was slightly prolonged, which is likely to be a reflection of its slow dissociation from CXCR428. A competition binding assay using a 125I-radiolabel on the CXCR4 monoclonal antibody 12G5 was also performed, which revealed that AMD3100 and AMD11070 were both unable to displace 12G5 on F292A CXCR4 (Figure 2G and H). This finding indicates that the efficacy switch for these two small molecules could be caused by either adoption of a different, non-competitive binding mode, or by a conformational effect on CXCR4 that alters the binding of 12G5. In fact, 12G5 binding to F292A was increased fourfold compared to WT CXCR4 (Bmax of 2030 ± 612 and 540 ± 183, respectively, Table S1). This was not due to differences in receptor surface expression (Figure 2B), which makes the conformational effect a more likely explanation. Together, the biological data for AMD3100 and AMD11070 suggests that these two compounds share some characteristics in their binding to CXCR4, leading to agonistic effect on F292A CXCR4. The binding mode for the bicyclam AMD3100 in CXCR4 has been extensively studied, and revealed that the drug positions itself across the major binding pocket29, with one cyclam ring interacting with Asp1714.60 in TM4 while the other cyclam ring sandwiches between Asp2626.58 and Glu2887.39 at the interface between TM6 and TM730, 31. Based on previously reported site-directed mutagenesis data32, a binding mode for AMD11070 that also involves Asp171 and Glu288 has been proposed by Cox et al.33. Thus, the literature suggests that AMD3100 and AMD11070 share a similar overall orientation in the binding pocket, connecting TM4 and TM7. To investigate how AMD3100 and AMD11070, but not the monocyclams, were switching efficacy, we constructed additional single mutants (D171N, D262N, and E288A) as well as a set of double mutants consisting of F292A plus alterations of the same three acidic residues. Furthermore, the single mutant H113A was included since His1133.29 has been shown to be an



Figure 2. Functional testing of the CXCR4 F292A mutation. (A) Ca2+ mobilization of 100 nM CXCL12 on WT and F292A CXCR4. Representative assays shown; n = 3. (B) Surface expression of WT and F292A CXCR4 receptors by ELISA; n = 13–16. (C+D) Gi signaling and inhibition of AMD3100 and AMD11070 on F292A CXCR4 by Gqi4myr-induced inositol phosphate (IP) accumulation with (white) and without (black) 10 nM CXCL12 treatment; n = 6– 9. WT shown as a stapled line; n = 21–24. (E+F) Ca2+ mobilization of 1 µM AMD3100 and AMD11070 on WT and F292A CXCR4. Representative assays shown; n = 3. (G+H) Competitive heterologous binding using 125I-12G5 and AMD3100 or AMD11070 on WT and F292A CXCR4; n = 6. All error bars presented as SEM.

interaction point for several CXCR4 antagonists 34 and this mutation has not previously been investigated for AMD11070. After confirming that all mutants were expressed at the cell surface (Figure S2), they were tested in G protein signaling (Figure 3A–D). Compared to the corresponding single mutations (without F292A; Figure 3A), the three double mutations (with F292A) resulted in a similar decrease in potency of CXCL12 (Figure 3B), where D262N and E288A had largest impact. For AMD3100, the single mutations impacting its antagonistic effect (Figure 3A) correlated with the F292A double mutants affecting its agonism (Figure 3C), following the same patterns as for CXCL12. Interestingly, the antagonism of AMD11070 on CXCL12 was not affected by alteration of either Asp residue (Figure 3A), but its agonistic potency on the F292A+D171N double mutant was almost 100-fold right shifted compared to F292A (Figure 3D). This finding shows that intrinsic activation of F292A CXCR4 by AMD11070 is dependent on Asp171, while this residue is not required for inhibition of CXCL12 signaling, which suggests somewhat different interaction patterns for its agonistic and antagonistic effect. Finally, the H113A mutation led to a 21-fold decrease in the antagonistic potency of AMD11070, while only slightly affecting AMD3100 (Figure 3A). To summarize, in F292A CXCR4 both ligands shared sites of importance for activation in Asp171 and Glu288, while Asp262 only impacted AMD3100. This finding is consistent with the suggested WT binding role for the cyclam ring in AMD3100 that is sandwiched between Asp262 and Glu28830, 31. Since AMD11070 lacks an equivalent structural element, this compound is not expected to show the same dual dependency in TM6/TM7, and thus only relies on Glu288. Furthermore, His113 was revealed to be of particular importance for the antagonistic effect of AMD11070. In an attempt to rationalize these findings on a molecular level, AMD11070 was docked35 to a receptor model of F292A CXCR4, which was generated from the CVX15-bound X-ray structure of CXCR4 (PDB code 3OE0)19. Several poses that involved Asp171 and Glu288, but not Asp262, were identified. In the top scoring pose (Figure 4A), Asp171 forms an ionic interaction with the primary amine while Glu288 is involved in a charge-assisted H-bond with the benzimidazole ring. These interactions are the same as for the WT binding model proposed by Cox et al.33; however, the orientations of the ring systems in AMD11070 differ significantly. Importantly, for F292A CXCR4 the benzimidazole moiety of AMD11070 goes deeper in the pocket and inserts into the space that is generated by the F292A mutation, i.e. a region that is occupied by the Phe292 phenyl ring in WT CXCR4. Here, the benzimidazole is surrounded by Tyr45/Phe87/Trp94/Tyr116, meaning that the aromatic “head” of AMD11070 to some extent takes over the role of Phe292 in the aromatic network (Figure 1B). Thus, it is possible for AMD11070 to latch on to Asp171 and Glu288 on each side of the major binding pocket of F292A CXCR4 while simultaneously engaging in the aromatic network in/around TM7. It is envisaged that the overall orientation of AMD3100 and AMD11070 in the binding pocket—where the ligands connect TM4 and TM7—in combination with the F292A mutation lead to partial receptor activation by affecting the behavior of TM7. In contrast, the monocyclams—which are unable to establish a similar connection between TM4 and TM734—remain unaffected by the F292A mutation. However, further experimental and computational studies are needed to fully establish the molecular details of the efficacy switch, and such studies are currently ongoing in our laboratory.


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Figure 3. Effect of CXCR4 mutations on CXCL12 inhibition and intrinsic activation by AMD3100 and AMD11070. (A) CXCL12 signaling and inhibition hereof by AMD3100 and AMD11070 on WT and mutated CXCR4 in Gqi4myr-induced IP accumulation. Data for CXCL12 and AMD3100 on H113A, D171N, D262N and E288A are reproductions 34 for direct comparison. (B–D) AMD3100 and AMD11070–induced activation of F292A containing CXCR4 double mutants in Gqi4myr-induced IP accumulation; n = 4–7. All error bars presented as SEM.

The agonism of AMD3100 and AMD11070 on F292A allowed us to establish a model for testing small molecule agonists as HIV fusion inhibitors. Thus, we performed a cell-cell fusion assay using “target cells” transfected with CD4, CXCR4 and firefly luciferase reporter, mixed with “effector cells” transfected with dual-tropic HIV Env and firefly luciferase activator. First, the fusion assay was validated by investigating the correlation between the aforementioned CXCR4 mutations’ effect on AMD3100 and AMD11070 in fusion inhibition and 12G5 displacement (Figure S3 and Table S1), as the affinity (obtained in competition binding with 125I-12G5) was previously suggested to be closely related to prevention of HIV entry36. In general, the fusion data showed good correlation with the binding data, thus validating the assay for the test. However, F292A was an exception due to the inability of AMD3100 and AMD11070 to displace 125I-12G5 on that mutant (Figure 2G and H). Next, AMD3100 and AMD11070 was tested as fusion inhibitors on F292A in parallel with WT CXCR4 (Figure 4B and C). The potencies of both AMD3100 (122 nM) and AMD11070 (145 nM) in fusion inhibition on F292A CXCR4 were not significantly different from their potencies on WT CXCR4 (89 nM and 52 nM, respectively). Furthermore, both compounds inhibited the fusion process to the same level on F292A and WT, suggesting that small molecule agonists can be powerful inhibitors of CXCR4-tropic HIV fusion. In summary, we here present a mutation (F292A) in CXCR4 that causes certain small molecule antagonists to gain agonistic properties without affecting their overall orientation in the binding pocket. This phenomenon is likely caused by an alteration of the aromatic network in/around TM7 (Tyr451.39/Trp942.60/Tyr1163.32/Trp2526.48/Phe2927.43) that allows AMD3100 and AMD11070 to initiate signaling in F292A CXCR4. We used this effect as a tool to show that small molecule agonists can function as strong inhibitors of HIV fusion. This finding matches our recent observation for CCR5 17, however, for CXCR4 it is of particular interest due to the inherent problems related to blocking the physiological functions of

CXCL12. While it is not very surprising that the small molecules are still able to inhibit fusion on F292A CXCR4, as they bind to the same region of the WT and mutant receptor, it

Figure 4. Docking of AMD11070 to F292A CXCR4, and testing of mutated receptor in fusion. (A) AMD11070 docked to the F292A CXCR4 structure; key interactions are highlighted by dotted lines: yellow = H-bonds, cyan = salt bridge, blue = - interactions, green = cation- interactions. (B+C) Inhibition of HIV gp120-mediated cell fusion by AMD3100 and AMD11070 on WT and F292A CXCR4; n = 5–7. All error bars presented as SEM.



emphasizes that discrimination between antagonists and agonists is of little importance when it comes to prevention of HIV entry. Furthermore, from a functional point of view, changing focus to development of small molecule CXCR4 agonists could prove favorable to prevent marked disruption of the CXCR4 system. Ideally, such agonist drugs should allow for receptor internalization, thus not provoking any WHIM-like unwanted effects. Recently, several novel small molecules targeting CXCR4 were reported18, including the first of their kind to display agonistic properties. These small molecule agonists could potentially be of use as anti-HIV drugs, however further studies are required to determine their potential.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures and supplementary figures and tables.

AUTHOR INFORMATION Corresponding Author * Phone: +45 30604608; E-mail: [email protected]

ORCID Christian Berg: orcid.org/0000-0002-0539-7081 Viktorija Daugvilaite: orcid.org/0000-0001-8786-348X Astrid S. Jørgensen: orcid.org/0000-0003-2736-8713

Present Addresses † Novo Nordisk A/S, Novo Allé, 2880 Bagsværd, Denmark

Author Contributions CB prepared the initial draft of the manuscript, and it was completed through contributions of all authors. MMR planned the project, and all authors participated in data analysis. CB, VD, AS and ASJ performed the in vitro experiments, and JV performed the molecular modeling and structural analysis. All authors have given approval to the final version of the manuscript.

Funding Sources This work was financially supported by the A.P. Møller Foundation, the Danish AIDS Foundation, the Thora and Viggo Grove Memorial Grant. V. Daugvilaite is furthermore supported by the Lundbeck Foundation.

Notes The authors declare no conflicts of interest.

ACKNOWLEDGMENT The authors wish to thank J. Sodroski, G. Bridger, M. Marsh, J. Hoxie, and E. Kostenis for providing plasmids and ligands. Furthermore, the authors wish to acknowledge O. Larsen for fruitful discussions and M. Pedersen for technical assistance. A special thanks to T. Schwartz for establishment of the initial CXCR4 library and his contributions to the project.

ABBREVIATIONS CXCR4, CXC chemokine receptor 4; CXCL12, CXC chemokine ligand 12; CCR5, CC chemokine receptor 5; HIV, human immunodefiency virus; HSC, hematopoietic stem cell; WHIM, Warts, Hypogammaglobulinaemia, Immunodeficiency, and Myelokathexis;

TM, transmembrane domain; WT, wild type; IP, inositol phosphate; ELISA, enzyme-linked immunosorbent assay;

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