**4. The development of CCR5 inhibitors as anti-HIV therapeutics – Focus on small molecules**

As discussed above, the first clues regarding the potential therapeutic utility of targeting CCR5 came from the observation of the naturally occurring blockade of the coreceptor due to the CCR5Δ32 polymorphism. Recently, the first successful allogeneic stem cell transplantation has been reported in an HIV-positive, acute myeloid leukemia patient from Berlin with a *CCR5*Δ*32* homozygous donor [63]. Investigators claimed to have "cured" this HIV-infected patient as indicated by the functional reconstitution of his T cell immunity without any signs of viral rebound after the discontinuation of ART [64]. The long-term effects of stem cell transplantation remain nevertheless unknown. Even though it would obviously be impractical to follow such a therapeutic approach in the millions of HIVinfected subjects worldwide, the proof-of-principle case of "the Berlin patient" demonstrates that a functional cure, i.e. a permanent suppression of viral replication in the absence of ART, may be within reach, under certain circumstances, at present; a true sterilizing cure with complete eradication of the virus continues to be the ultimate goal of therapy [6].

Towards this goal, there has been hope that CCR5 inhibitors will make an impact on controlling the epidemic by enhancing our therapeutic arsenal against HIV.

Chemokine Receptors as Therapeutic Targets in HIV Infection 123

HIV mechanism of native chemokines. Further N-terminal modifications led to the generation of chemokine analogues such as AOP-RANTES [73] and PSC-RANTES [74], which have improved potency due to their mechanism of action; they induce receptor sequestration that results in the internalization and physical removal of the coreceptor from the cell surface, effects that are more profound and prolonged compared to those of native chemokines [22]. The clinical development of chemokine analogues as anti-HIV therapeutics is complicated by the fact that as proteins, chemokines would not be expected to be orally bioavailable and their production costs would be prohibitively high; moreover, chemokine analogues are unlikely to show suitable pharmacokinetics after injection because they readily bind to and form aggregates on cell surface proteoglycans [22]. The potential adverse effects due to the possible inflammatory activity mediated through CCR5 activation is

Most anti-CCR5 monoclonal antibodies (MAbs) have been described as chemokine antagonists that inhibit the receptor via steric blockade (reviewed in [22]). Many rodent anti-CCR5 MAbs with anti-HIV activity have been identified and extensively characterized. PRO 140 (Progenics pharmaceuticals) is a humanized monoclonal antibody, currently available only for parenteral administration; PRO 140 received fast-track approval by the FDA. The efficacy, tolerability and toxicity profiles of PRO 140 have been assessed by several preclinical and clinical studies, which showed promising results (reviewed in [75]). Of note is the evidence of activity shown by PRO 140 against escape mutants with cross-class resistance to small molecule CCR5 inhibitors [76]; however, as a complex biological molecule requiring parenteral administration, PRO 140 faces a different set of development challenges compared to the orally bioavailable small molecule compounds [77]. Human CCR5mAb004 antibody also performed well in early clinical trials [22]. Currently available data further suggest that anti-CCR5 MAbs act synergistically with other CCR5 inhibitors and have different resistance profiles. Their typically long circulatory half-lives do not necessitate frequent dosing, suggesting that the lack of oral bioavailability may not be a

CCR5 expression in HIV-1 target cells can be suppressed by RNA-based gene modulation technologies such as RNA interference (RNAi), or completely eliminated by such techniques as zinc finger nuclease (ZFN)-mediated gene disruption. RNAi refers to the sequence-specific degradation of messenger RNA (mRNA) that follows the cellular introduction of homologous, short-interfering RNA (siRNA), a technique that has emerged as a powerful tool to probe the function of genes of known sequence *in vitro* and *in vivo* [78, 79]. RNAi has been shown to decrease the replication of HIV-1 in lymphocytic cells using siRNA targeting both viral (e.g., Tat, Gag and Rev) and host proteins, including CD4 and CCR5 (reviewed in [80]). The numerous challenges associated with converting RNAi from a laboratory technique to an anti-HIV therapeutic have been

another concern associated with their use.

serious limitation in their development as anti-HIV drugs.

**4.3. Gene knockdown and knockout strategies** 

**4.2. Anti-CCR5 antibodies** 

It should be noted, however, that the development of these host-targeting agents raises additional safety concerns that lie beyond the typical pharmacological concerns associated with the development of any antiretroviral drug, such as efficacy, safety and tolerability, bioavailability, drug-drug interactions, and the emergence of escape mutants (reviewed in [22, 65]). These safety concerns stem from the disruption of the physiological functions of the coreceptor as well as from the potential escape of R5 viruses by switching coreceptor use to CXCR4, or the breakthrough of X4 viruses following the suppression of R5 strains [22, 65]. The latter virological concern will be discussed in the next section on the generation of resistance to CCR5 inhibitors. The absence of any apparent immunological deficits of significance among individuals with naturally occurring loss-of-function mutations (i.e., *CCR5*Δ*32* homozygotes) provides some reassurance that pharmacological blockade of CCR5 will be relatively benign [66]. Redundancy in the chemokine network presumably allows other chemokine receptors to subsume the function of CCR5 [67]. However, the abrupt pharmacological blockade of a receptor in mature individuals may have different consequences than the congenital absence of the receptor where the immune system has had years to adapt to its loss [22, 67]. Corroborating the results of previous studies in mice (reviewed in [67]), studies in humans have shown that *CCR5∆32* heterozygotes have a sixfold increased risk for severe West Nile virus (WNV) infection and a five-fold increased risk of mortality [68]. A meta-analysis of the four patient cohorts in the United States confirmed that CCR5 deficiency is a strong and consistent risk factor for symptomatic WNV infection [69]. An additional association between the lack of functional CCR5 due to the CCR5∆32 deletion and tick-borne encephalitis has also been reported [70]. Overall, however, currently available data suggest that pharmacological blockade of CCR5 is likely to be largely well tolerated; stringent safety studies will nevertheless be required to monitor the long-term safety of CCR5 blockade, with particular attention paid to the consequences of infection by certain pathogens, effects on responses to immunization, and the emergence of opportunistic infections and malignancies [22, 65].

Several different approaches have been undertaken to pharmaceutically target CCR5, including the use of chemokine analogues, anti-CCR5 antibodies, gene knockdown and knockout strategies, and small molecule coreceptor inhibitors. The development of coreceptor inhibitors for use in prevention, including topical microbicides, is discussed elsewhere [22, 67].

#### **4.1. Chemokine analogues**

Chemokine analogues engineered to enhance their natural anti-HIV properties were the first generation of CCR5 inhibitors (reviewed in [22, 71, 72]). RANTES, an N-terminally truncated variant of RANTES/CCL5 was the first such engineered analogue, but it was found to exhibit only modest anti-HIV activity. Its identification nonetheless helped establish the concept that the agonist activity on coreceptors is not a component of the antiHIV mechanism of native chemokines. Further N-terminal modifications led to the generation of chemokine analogues such as AOP-RANTES [73] and PSC-RANTES [74], which have improved potency due to their mechanism of action; they induce receptor sequestration that results in the internalization and physical removal of the coreceptor from the cell surface, effects that are more profound and prolonged compared to those of native chemokines [22]. The clinical development of chemokine analogues as anti-HIV therapeutics is complicated by the fact that as proteins, chemokines would not be expected to be orally bioavailable and their production costs would be prohibitively high; moreover, chemokine analogues are unlikely to show suitable pharmacokinetics after injection because they readily bind to and form aggregates on cell surface proteoglycans [22]. The potential adverse effects due to the possible inflammatory activity mediated through CCR5 activation is another concern associated with their use.

#### **4.2. Anti-CCR5 antibodies**

122 Immunodeficiency

*CCR5*Δ

Towards this goal, there has been hope that CCR5 inhibitors will make an impact on

It should be noted, however, that the development of these host-targeting agents raises additional safety concerns that lie beyond the typical pharmacological concerns associated with the development of any antiretroviral drug, such as efficacy, safety and tolerability, bioavailability, drug-drug interactions, and the emergence of escape mutants (reviewed in [22, 65]). These safety concerns stem from the disruption of the physiological functions of the coreceptor as well as from the potential escape of R5 viruses by switching coreceptor use to CXCR4, or the breakthrough of X4 viruses following the suppression of R5 strains [22, 65]. The latter virological concern will be discussed in the next section on the generation of resistance to CCR5 inhibitors. The absence of any apparent immunological deficits of significance among individuals with naturally occurring loss-of-function mutations (i.e.,

*32* homozygotes) provides some reassurance that pharmacological blockade of CCR5 will be relatively benign [66]. Redundancy in the chemokine network presumably allows other chemokine receptors to subsume the function of CCR5 [67]. However, the abrupt pharmacological blockade of a receptor in mature individuals may have different consequences than the congenital absence of the receptor where the immune system has had years to adapt to its loss [22, 67]. Corroborating the results of previous studies in mice (reviewed in [67]), studies in humans have shown that *CCR5∆32* heterozygotes have a sixfold increased risk for severe West Nile virus (WNV) infection and a five-fold increased risk of mortality [68]. A meta-analysis of the four patient cohorts in the United States confirmed that CCR5 deficiency is a strong and consistent risk factor for symptomatic WNV infection [69]. An additional association between the lack of functional CCR5 due to the CCR5∆32 deletion and tick-borne encephalitis has also been reported [70]. Overall, however, currently available data suggest that pharmacological blockade of CCR5 is likely to be largely well tolerated; stringent safety studies will nevertheless be required to monitor the long-term safety of CCR5 blockade, with particular attention paid to the consequences of infection by certain pathogens, effects on responses to immunization, and the emergence of

Several different approaches have been undertaken to pharmaceutically target CCR5, including the use of chemokine analogues, anti-CCR5 antibodies, gene knockdown and knockout strategies, and small molecule coreceptor inhibitors. The development of coreceptor inhibitors for use in prevention, including topical microbicides, is discussed

Chemokine analogues engineered to enhance their natural anti-HIV properties were the first generation of CCR5 inhibitors (reviewed in [22, 71, 72]). RANTES, an N-terminally truncated variant of RANTES/CCL5 was the first such engineered analogue, but it was found to exhibit only modest anti-HIV activity. Its identification nonetheless helped establish the concept that the agonist activity on coreceptors is not a component of the anti-

controlling the epidemic by enhancing our therapeutic arsenal against HIV.

opportunistic infections and malignancies [22, 65].

elsewhere [22, 67].

**4.1. Chemokine analogues** 

Most anti-CCR5 monoclonal antibodies (MAbs) have been described as chemokine antagonists that inhibit the receptor via steric blockade (reviewed in [22]). Many rodent anti-CCR5 MAbs with anti-HIV activity have been identified and extensively characterized. PRO 140 (Progenics pharmaceuticals) is a humanized monoclonal antibody, currently available only for parenteral administration; PRO 140 received fast-track approval by the FDA. The efficacy, tolerability and toxicity profiles of PRO 140 have been assessed by several preclinical and clinical studies, which showed promising results (reviewed in [75]). Of note is the evidence of activity shown by PRO 140 against escape mutants with cross-class resistance to small molecule CCR5 inhibitors [76]; however, as a complex biological molecule requiring parenteral administration, PRO 140 faces a different set of development challenges compared to the orally bioavailable small molecule compounds [77]. Human CCR5mAb004 antibody also performed well in early clinical trials [22]. Currently available data further suggest that anti-CCR5 MAbs act synergistically with other CCR5 inhibitors and have different resistance profiles. Their typically long circulatory half-lives do not necessitate frequent dosing, suggesting that the lack of oral bioavailability may not be a serious limitation in their development as anti-HIV drugs.

#### **4.3. Gene knockdown and knockout strategies**

CCR5 expression in HIV-1 target cells can be suppressed by RNA-based gene modulation technologies such as RNA interference (RNAi), or completely eliminated by such techniques as zinc finger nuclease (ZFN)-mediated gene disruption. RNAi refers to the sequence-specific degradation of messenger RNA (mRNA) that follows the cellular introduction of homologous, short-interfering RNA (siRNA), a technique that has emerged as a powerful tool to probe the function of genes of known sequence *in vitro* and *in vivo* [78, 79]. RNAi has been shown to decrease the replication of HIV-1 in lymphocytic cells using siRNA targeting both viral (e.g., Tat, Gag and Rev) and host proteins, including CD4 and CCR5 (reviewed in [80]). The numerous challenges associated with converting RNAi from a laboratory technique to an anti-HIV therapeutic have been

reviewed elsewhere [22]. Recent advances in the development of RNAi-based therapeutics for HIV-1 are presented by Zhou and Rossi [81]. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes engineered by fusing a zinc finger DNA-binding domain to a DNAcleavage domain; the generated double-stranded DNA break is repaired by the endogenous DNA repair machinery of the cell, potentially leading to the permanent disruption of the gene's open reading frame [82]. CCR5 gene knockout in T cells or hematopoietic stem/progenitor cells (HSC) by ZFNs effectively suppresses the replication of CCR5-tropic strains of HIV-1 in animal models (reviewed in [83]). ZFNs are currently being evaluated in a phase I clinical trials using *ex vivo* expanded T cells; HSC targeted therapies are under development as well.

Chemokine Receptors as Therapeutic Targets in HIV Infection 125

Takeda Preclinical

Takeda; now Tobira

Schering-Plough

Schering-Plough

Schering-Plough; now Merck

Glaxo SmithKline **status** 

only

Phase II completed

Preclinical only

Discontinued; hERG K+ channel blockade & CNS side effects

Discontinued from Phase III; primary efficacy points unmet

Discontinued from Phase IIb/III; idiosyncra tic hepatotoxi city

for clinical use

Pfizer Approved

**References** 

[84]

[85, 86]

[87, 88]

[90-92]

[93, 94]

[95]

[96, 97]

**Drug Other names Key features Manufacturer Current** 

ammonium anilide that prevents gp120 and CCchemokines, but not anti-CCR5 MAbs, from binding to CCR5

also blocks CCR2

macaques [89]

functions

UK-427,857 Spirodiketopiperazine with

properties

Orally bioavailable, improved potency TAK-779-derivative;

compound structurally related to, but more potent than SCH-C; also blocks MAbs mainly against the ECL2 of CCR5

amide that also blocks MAbs mainly against the ECL2 of CCR5; unable to exert its antiviral effects when residue 198 in TM helix 5 of CCR5 is methionine (Met) as in rhesus

Piperazino-piperidine-based compound; improved potency & pharmacokinetics, reduced tendency for hERG channel blockade compared to SCH-C

Spirodiketopiperazine-based compound; it preserves RANTES and MIP-1β binding to CCR5+ cells and their

favorable pharmacological

**hERG K+**, human ether-a-go-go related gene potassium channel; **MAb**, monoclonal antibody; **N/A**, not applicable.

**Table 1.** Nomenclature, characteristics, and developmental status of selected small molecule CCR5

TAK-779 N/A (Nonpeptidic) quaternary

AD101 SCH-350581 Piperidino-piperazine-based

SCH-C SCH-351125 Oximino-piperidino-piperidine

Cenicriviroc TBR-652,

Vicriviroc (VCV)

Aplaviroc (AVC)

Maraviroc (MVC, Selzentry)

inhibitors

TAK-652

SCH-D, SCH-417690, VVC

AK602/ONO41 28/873140, GW873140, APL

#### **4.4. Small molecule CCR5 inhibitors**

Small molecule CCR5 inhibitors (Table 1) prevent HIV-1 from entering its target cells, mainly CD4+ T cells and macrophages, by blocking the CD4-dependent binding of the viral surface envelope glycoprotein (gp120) to the most commonly used coreceptor CCR5 (reviewed in [22, 67, 72, 77]).

The identification of the first such molecule, TAK-779, in 1999 [84] was followed by several reports on additional CCR5 inhibitors with improved potency and more favorable pharmacological properties (Table 1). The development of these compounds has been principally pursued for the treatment of HIV-1 infection, although, theoretically, they could also be used as preventive agents [98]. Maraviroc (MVC, Selzentry; Pfizer, Inc., Figure 2A) was the first, and so far only, small molecule CCR5 inhibitor to be approved for treating HIV-1 infection by the US FDA and by the European Agency for the Evaluation of Medicinal Products (reviewed in [99]). MVC is licensed for administration in both treatment-naïve (i.e., yet to receive any ART) and in treatment-experienced patients with R5 HIV-1. Other investigational compounds have also entered clinical trials, but a number of them were discontinued due to observed side effects (Table 1). The development of Vicriviroc (VCV, Schering-Plough Research Institute; now Merck Research Laboratories, Figure 2B) was abandoned because it did not meet primary efficacy endpoints in late stage (Phase III in treatment-experienced subjects and Phase II in treatment-naïve individuals) clinical trials. Additional compounds are in preclinical or clinical development (reviewed in [77]). Previously designated as TBR-652 or TAK-652 (Figure 2C), cenicriviroc is a small-molecule CCR5 antagonist licensed by the Takeda Pharmaceutical Company to Tobira Pharmaceuticals; cenicriviroc has pharmacokinetic data supportive of once daily dosing and a favorable tolerability profile in single-dose studies in healthy volunteers. PF-232798 (Figure 2D), a second-generation small-molecule oral CCR5 antagonist with potency similar to MVC and the potential for once daily dosing, has demonstrated activity against a laboratory-generated MVC-resistant R5 virus. INCB9471 (Incyte, Wilmington, DE, USA) is yet another small-molecule oral CCR5 inhibitor that showed promising results in a Phase IIa trial in treatment-naïve and treatment-experienced patients with once daily dosing over 14 days [77].


therapies are under development as well.

**4.4. Small molecule CCR5 inhibitors** 

(reviewed in [22, 67, 72, 77]).

days [77].

reviewed elsewhere [22]. Recent advances in the development of RNAi-based therapeutics for HIV-1 are presented by Zhou and Rossi [81]. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes engineered by fusing a zinc finger DNA-binding domain to a DNAcleavage domain; the generated double-stranded DNA break is repaired by the endogenous DNA repair machinery of the cell, potentially leading to the permanent disruption of the gene's open reading frame [82]. CCR5 gene knockout in T cells or hematopoietic stem/progenitor cells (HSC) by ZFNs effectively suppresses the replication of CCR5-tropic strains of HIV-1 in animal models (reviewed in [83]). ZFNs are currently being evaluated in a phase I clinical trials using *ex vivo* expanded T cells; HSC targeted

Small molecule CCR5 inhibitors (Table 1) prevent HIV-1 from entering its target cells, mainly CD4+ T cells and macrophages, by blocking the CD4-dependent binding of the viral surface envelope glycoprotein (gp120) to the most commonly used coreceptor CCR5

The identification of the first such molecule, TAK-779, in 1999 [84] was followed by several reports on additional CCR5 inhibitors with improved potency and more favorable pharmacological properties (Table 1). The development of these compounds has been principally pursued for the treatment of HIV-1 infection, although, theoretically, they could also be used as preventive agents [98]. Maraviroc (MVC, Selzentry; Pfizer, Inc., Figure 2A) was the first, and so far only, small molecule CCR5 inhibitor to be approved for treating HIV-1 infection by the US FDA and by the European Agency for the Evaluation of Medicinal Products (reviewed in [99]). MVC is licensed for administration in both treatment-naïve (i.e., yet to receive any ART) and in treatment-experienced patients with R5 HIV-1. Other investigational compounds have also entered clinical trials, but a number of them were discontinued due to observed side effects (Table 1). The development of Vicriviroc (VCV, Schering-Plough Research Institute; now Merck Research Laboratories, Figure 2B) was abandoned because it did not meet primary efficacy endpoints in late stage (Phase III in treatment-experienced subjects and Phase II in treatment-naïve individuals) clinical trials. Additional compounds are in preclinical or clinical development (reviewed in [77]). Previously designated as TBR-652 or TAK-652 (Figure 2C), cenicriviroc is a small-molecule CCR5 antagonist licensed by the Takeda Pharmaceutical Company to Tobira Pharmaceuticals; cenicriviroc has pharmacokinetic data supportive of once daily dosing and a favorable tolerability profile in single-dose studies in healthy volunteers. PF-232798 (Figure 2D), a second-generation small-molecule oral CCR5 antagonist with potency similar to MVC and the potential for once daily dosing, has demonstrated activity against a laboratory-generated MVC-resistant R5 virus. INCB9471 (Incyte, Wilmington, DE, USA) is yet another small-molecule oral CCR5 inhibitor that showed promising results in a Phase IIa trial in treatment-naïve and treatment-experienced patients with once daily dosing over 14

**hERG K+**, human ether-a-go-go related gene potassium channel; **MAb**, monoclonal antibody; **N/A**, not applicable.

**Table 1.** Nomenclature, characteristics, and developmental status of selected small molecule CCR5 inhibitors

Chemokine Receptors as Therapeutic Targets in HIV Infection 127

sensitivity to SCH-C and AVC [110]. Susceptibility to other classes of licensed drugs that target different stages of the viral life cycle, or even different steps of the same stage of the viral life cycle such as viral entry, should be maintained. Accordingly, clinical resistance to Enfuvirtide (ENF, Fuzeon/T-20) was not found to affect viral sensitivity to the fusion

**Mechanism of action.** Small molecule (molecular weight 500-600 D) CCR5 inhibitors act via a similar, allosteric (from the Greek "*allos*"+"*stereos*" meaning "other"+"shape," respectively) mechanism; in other words, they induce or stabilize a conformation of the coreceptor that renders it unrecognizable by the wild-type virus. The results of recent biochemical studies support the noncompetitive and allosteric nature of the mechanism of action of small molecule antagonists of CCR5 [104]. The difference in binding sites between the two ligands also supports the allosteric modulation of the HIV-1 gp120 binding site by this class of drugs; as mentioned above, CCR5 inhibitors bind within the TM helices in the extracellular facet of the coreceptor, whereas HIV gp120 targets the extracellular domains of CCR5 and, more specifically, the negatively-charged Tyr-sulfated amino terminus (Nt) and the ECL2

Moreover, the action of some CCR5 inhibitors may be blocked by the introduction of certain mutations into CCR5 despite the binding of the inhibitor to the coreceptor; namely, the antiviral effects of SCH-C are blocked, although its antagonism of the signaling by RANTES/CCL5 persists, when residue 198 in the TM helix 5 of CCR5 is methionine (Met) as in rhesus macaques [89]. This observation led to the hypothesis that the I198M substitution may be involved in the induction of a CCR5 conformation that is incompatible with HIV-1 infection. It should be noted, however, that not all CCR5 inhibitors are modulated in the same manner by this amino acid change. I198M has been shown not to block the antiviral effects of AD101, whereas another mutation, F113A, may block the inhibitory action of AD101, but not of SCH-C, via a similar mechanism [89]. Further subtle differences exist in the stabilized or induced conformations by the binding of these small molecules to the TM pocket of CCR5 and, therefore, in the mechanism of action of these compounds. For instance, although most CCR5 inhibitors antagonize potently the three natural ligands of the coreceptor, AVC only antagonizes MIP-1α/CCL3 efficiently and much less potently the

Displacement binding assays and dissociation kinetics demonstrated that TAK779 and MVC inhibit CCL3 and gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that these compounds bind to regions of CCR5 distinct from the gp120 and CCL3-binding sites [112]. MVC is predicted to insert deeply in the CCR5 TM cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct, yet overlapped regions of the CCR5 ECL2 [113]. TAK779 and MVC were found to be full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins [112]. The finding that the TM cavity remains accessible for MVC in CCL3-bound and gp120-bound CCR5 [113], provides an explanation for the enhancement by TAK779 and MVC of the dissociation of

inhibitor T-1249 or to coreceptor inhibitors [111].

other two ligands (MIP-1β/CCL4, and RANTES/CCL5) [95].

between TM helices 4 and 5 [103].

**Figure 2.** Chemical structures of selected small molecule CCR5 inhibitors. A. Maraviroc (MVC, Selzentry), B. Vicriviroc (VCV), C. Cenicriviroc (TBR-652), D. PF-232798.

**Binding site.** The binding site of small molecule CCR5 inhibitors does not overlap with the binding site for HIV gp120. In the absence of a high-resolution structure of a gp120 coreceptor complex, biochemical studies of CCR5 have revealed the importance of its Nterminus (Nt) and second extracellular loop (ECL2) in binding gp120 and mediating viral entry (reviewed in [34]). Small molecule CCR5 inhibitors, on the other hand, are believed to bind within a cavity between the transmembrane (TM) helices on the extracellular surface of the coreceptor, as suggested by site-directed mutagenesis experiments and molecular modeling of CCR5 on the basis of the available crystal structure of bovine rhodopsin that is characterized by a similar TM topology [100, 101]. More specifically, the binding "pocket" of such molecules as TAK-779, AD101, and SCH-C is thought to be located between TM helices 1-3, and 7; other TM helices, particularly helix 6, contribute to some compound-specific variations [102, 103]. Simultaneous administration of multiple antagonists showed that, indeed, they bind to a common site on CCR5 [104]. A key set of aromatic and aliphatic residues that serve as a hydrophobic core for this ligand binding pocket has also been identified, while a glutamic acid (Glu) in helix 7, E283, appears to be critical for high affinity interaction; this residue probably acts as the counterion for a positively charged nitrogen atom common to TAK-779, AD101, and SCH-C [105].

The binding of all small molecule CCR5 antagonists to a broadly similar site within the TM helices of the coreceptor renders cross-resistance within this drug class possible, but not necessarily inevitable since compound-specific variations with respect to properties such as structure, shape, and electrostatic potential, do exist [106]; hence, despite the insensitivity of AD101 escape mutants to all other tested CCR5 inhibitors, including SCH-C, aplaviroc (AVC), MVC and VCV [107-109], a MVC-resistant variant has been reported to retain its sensitivity to SCH-C and AVC [110]. Susceptibility to other classes of licensed drugs that target different stages of the viral life cycle, or even different steps of the same stage of the viral life cycle such as viral entry, should be maintained. Accordingly, clinical resistance to Enfuvirtide (ENF, Fuzeon/T-20) was not found to affect viral sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors [111].

126 Immunodeficiency

**Figure 2.** Chemical structures of selected small molecule CCR5 inhibitors. A. Maraviroc (MVC,

**Binding site.** The binding site of small molecule CCR5 inhibitors does not overlap with the binding site for HIV gp120. In the absence of a high-resolution structure of a gp120 coreceptor complex, biochemical studies of CCR5 have revealed the importance of its Nterminus (Nt) and second extracellular loop (ECL2) in binding gp120 and mediating viral entry (reviewed in [34]). Small molecule CCR5 inhibitors, on the other hand, are believed to bind within a cavity between the transmembrane (TM) helices on the extracellular surface of the coreceptor, as suggested by site-directed mutagenesis experiments and molecular modeling of CCR5 on the basis of the available crystal structure of bovine rhodopsin that is characterized by a similar TM topology [100, 101]. More specifically, the binding "pocket" of such molecules as TAK-779, AD101, and SCH-C is thought to be located between TM helices 1-3, and 7; other TM helices, particularly helix 6, contribute to some compound-specific variations [102, 103]. Simultaneous administration of multiple antagonists showed that, indeed, they bind to a common site on CCR5 [104]. A key set of aromatic and aliphatic residues that serve as a hydrophobic core for this ligand binding pocket has also been identified, while a glutamic acid (Glu) in helix 7, E283, appears to be critical for high affinity interaction; this residue probably acts as the counterion for a positively charged nitrogen

The binding of all small molecule CCR5 antagonists to a broadly similar site within the TM helices of the coreceptor renders cross-resistance within this drug class possible, but not necessarily inevitable since compound-specific variations with respect to properties such as structure, shape, and electrostatic potential, do exist [106]; hence, despite the insensitivity of AD101 escape mutants to all other tested CCR5 inhibitors, including SCH-C, aplaviroc (AVC), MVC and VCV [107-109], a MVC-resistant variant has been reported to retain its

Selzentry), B. Vicriviroc (VCV), C. Cenicriviroc (TBR-652), D. PF-232798.

atom common to TAK-779, AD101, and SCH-C [105].

**Mechanism of action.** Small molecule (molecular weight 500-600 D) CCR5 inhibitors act via a similar, allosteric (from the Greek "*allos*"+"*stereos*" meaning "other"+"shape," respectively) mechanism; in other words, they induce or stabilize a conformation of the coreceptor that renders it unrecognizable by the wild-type virus. The results of recent biochemical studies support the noncompetitive and allosteric nature of the mechanism of action of small molecule antagonists of CCR5 [104]. The difference in binding sites between the two ligands also supports the allosteric modulation of the HIV-1 gp120 binding site by this class of drugs; as mentioned above, CCR5 inhibitors bind within the TM helices in the extracellular facet of the coreceptor, whereas HIV gp120 targets the extracellular domains of CCR5 and, more specifically, the negatively-charged Tyr-sulfated amino terminus (Nt) and the ECL2 between TM helices 4 and 5 [103].

Moreover, the action of some CCR5 inhibitors may be blocked by the introduction of certain mutations into CCR5 despite the binding of the inhibitor to the coreceptor; namely, the antiviral effects of SCH-C are blocked, although its antagonism of the signaling by RANTES/CCL5 persists, when residue 198 in the TM helix 5 of CCR5 is methionine (Met) as in rhesus macaques [89]. This observation led to the hypothesis that the I198M substitution may be involved in the induction of a CCR5 conformation that is incompatible with HIV-1 infection. It should be noted, however, that not all CCR5 inhibitors are modulated in the same manner by this amino acid change. I198M has been shown not to block the antiviral effects of AD101, whereas another mutation, F113A, may block the inhibitory action of AD101, but not of SCH-C, via a similar mechanism [89]. Further subtle differences exist in the stabilized or induced conformations by the binding of these small molecules to the TM pocket of CCR5 and, therefore, in the mechanism of action of these compounds. For instance, although most CCR5 inhibitors antagonize potently the three natural ligands of the coreceptor, AVC only antagonizes MIP-1α/CCL3 efficiently and much less potently the other two ligands (MIP-1β/CCL4, and RANTES/CCL5) [95].

Displacement binding assays and dissociation kinetics demonstrated that TAK779 and MVC inhibit CCL3 and gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that these compounds bind to regions of CCR5 distinct from the gp120 and CCL3-binding sites [112]. MVC is predicted to insert deeply in the CCR5 TM cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct, yet overlapped regions of the CCR5 ECL2 [113]. TAK779 and MVC were found to be full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins [112]. The finding that the TM cavity remains accessible for MVC in CCL3-bound and gp120-bound CCR5 [113], provides an explanation for the enhancement by TAK779 and MVC of the dissociation of preformed ligand-CCR5 complexes with an efficiency that correlates with their ability to act as inverse agonists [112]. The identification of residues mandatory for gp120 binding in the predicted CCR5 dimer interface suggests that receptor dimerization might represent a target for new CCR5 entry inhibitors [113].

Chemokine Receptors as Therapeutic Targets in HIV Infection 129

**5.1.** *In vitro* **resistance: No coreceptor switch to CXCR4 and no uniform** 

conditions, which may not be very relevant physiologically [120].

[124, 126, 132], but that is not always the case [129].

To gain insights into what may happen *in vivo* if or when small molecule CCR5 inhibitors are used more extensively, resistance is first studied *in vitro*. To generate resistant variants, primary R5 isolates are cultured with physiological target cells that are permissive for replication of both R5 and X4 viruses in the presence of increasing concentrations of the inhibitor under study. Despite the intrinsic difficulty associated with the selection process in each case that proved to require prolonged periods of culture in the presence of these compounds, several such studies have been published (reviewed in [22, 72, 117]). An alternative *in vitro* system that allows for the selection of escape mutants to CCR5 inhibitors over a relatively short period of time is the construction of V3 loop libraries of R5 infectious clones (reviewed in [118]). The results of these studies collectively show that the escape mutants tend to retain usage of CCR5 despite the availability of CXCR4. The mechanism that allows resistant viruses to continue to use CCR5 in the presence of the inhibitor is discussed in the next section. X4 viruses have been observed in CCR5 inhibitor-selection experiments, either in the control culture alone [119] or in both the selection and the control cultures [110], but their emergence was probably due to adaptation to growth in culture rather than the inhibitor escape process. Conditional recognition of CXCR4 in specific cell lines has also been reported for one VCV-resistant virus [109]. HIV-1 can be "forced" to escape CCR5 drug pressure through coreceptor switch under certain experimental

To date, no clear signature resistance mutations have been identified for CCR5 antagonists

The determinants of resistance commonly map to the V3 region of gp120, a localization that is consistent with the highly variable nature of this region as well as with its role in coreceptor engagement [22, 45]. Four V3 substitutions (K305R, H308P, A316V and G321E) were, for example, responsible for the resistant phenotype of isolate CC101.19 when the CCR5-using, subtype B virus CC1/85 [62] was cultured with the small molecule CCR5 inhibitor AD101, a preclinical precursor of VCV [107, 108]. A much rarer route to resistance was undertaken by viruses derived from the same CC1/85 lineage in an AD101/VCV selection culture [109]; the resulting D101.12 escape mutants contained at least one substitution in V3 (H308P) as well as either of the following combinations of substitutions within and downstream of the gp41 fusion peptide (FP): G514V+V535M (Pattern I) or M518V+F519L+V535M (Pattern II) [127]. We have also described a unique VCV escape pathway for viruses from the D1/85.16 lineage that involved no V3 changes; instead, resistance mapped to three conservative substitutions in the FP: G516V, M518V and F519I [126]. As shown recently [131], the G516V change is critical to VCV resistance, although it must be accompanied by either M518V or F519I to have a substantial impact phenotypically. Nevertheless, introducing the three FP changes together into a heterologous virus, JR-FL, did not create a VCV-resistant variant [126]. CCR5 inhibitor resistance is usually dependent upon the Env genetic context and it is not transferred when introduced into other viruses

and different genetic pathways can lead to the same resistance phenotype (Table 2).

**genotypic signature** 

AK530 and AK317, two CCR5 inhibitors containing spirodiketopiperazine scaffolds, lodge in a hydrophobic cavity located between the upper TM domain and the ECL2 of CCR5 [114]. The interaction profile of the inhibitors with ECL2 residues S180 and K191 was found to be an important determinant of antiviral potency; furthermore, amino acid residues in the betahairpin structural motif of ECL2 were critical for HIV-1-elicited fusion and binding of these spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but it also prohibits the virus from utilizing the "inhibitor-bound" CCR5 for cellular entry - a mechanism of HIV-1's resistance to CCR5 inhibitors, as discussed in the next section.
