**2. The HIV entry process and the coreceptors**

#### **2.1. Discovery of the HIV coreceptors**

CD4 had been known to be the principal cell surface receptor for HIV-1, and also for HIV-2 and simian immunodeficiency virus (SIV), since 1984 [7, 8]. It had also been known for almost as long, however, that the presence of the CD4 antigen alone was not sufficient to allow for HIV entry. To infect CD4+ cells, HIV-1 strains of different biological phenotypes, namely macrophage (M)-tropic and T-cell line (T)-tropic, were supposed to utilize different auxiliary co-factors [9], the identity of which remained elusive until early 1996. At that time, a member of the seven-transmembrane (7-TM) spanning family of chemokine receptors initially termed "fusin", was identified as the coreceptor for T-tropic HIV-1 variants [10]; the term was changed to C-X-C chemokine receptor type 4 (CXCR4) once its natural ligand was discovered to be stromal derived factor-1 (SDF-1)/CXCL12 [11, 12]. CXCR4 had been previously identified as an orphan receptor called leukocyte-derived seven-transmembrane domain receptor (LESTR), but it only received attention after its isolation as an HIV-1 coreceptor [13].

The knowledge that the β-chemokines macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4 and regulated upon activation normal T-cell expressed and secreted (RANTES)/CCL5 exhibited antiviral activity against M-tropic HIV-1 isolates *in vitro* [14], provided the basis for the discovery of C-C chemokine receptor type 5 (CCR5) as the entry co-factor for these strains by five groups simultaneously [15-19]. The virus thus uses chemokine receptors, mainly CCR5 and CXCR4, to enter susceptible cells, while the cognate chemokine ligands act as natural entry inhibitors. Other chemokine receptors may also function as HIV coreceptors; nonetheless, infection of primary cells via alternative coreceptors is rare and the process can normally be fully blocked by CCR5 or CXCR4 inhibitors [20-22]. Hence, at present, CCR5 and CXCR4 are considered as the only HIV coreceptors of physiological significance.

#### **2.2. Physiological roles of the main coreceptors**

116 Immunodeficiency

currently limited by chronic toxicities or by the presence of resistance to conventional

This chapter focuses on describing the scientific rationale that led to the development of coreceptor inhibitors as a new class of host-targeted antiretroviral agents. This description entails a revision of the biology of coreceptor usage during HIV entry into target cells and viral tropism during the course of infection. The various approaches undertaken to pharmaceutically target the most commonly used coreceptor, CCR5, will then be described, with a special focus on small molecule CCR5 inhibitors (also termed "CCR5 antagonists") and their mechanism of action. The current status of the small molecule CCR5 inhibitor pipeline, with emphasis on the generation of resistance, including *in vitro* and *in vivo* HIV escape pathways and mechanisms, will be presented, while implications as well as future

CD4 had been known to be the principal cell surface receptor for HIV-1, and also for HIV-2 and simian immunodeficiency virus (SIV), since 1984 [7, 8]. It had also been known for almost as long, however, that the presence of the CD4 antigen alone was not sufficient to allow for HIV entry. To infect CD4+ cells, HIV-1 strains of different biological phenotypes, namely macrophage (M)-tropic and T-cell line (T)-tropic, were supposed to utilize different auxiliary co-factors [9], the identity of which remained elusive until early 1996. At that time, a member of the seven-transmembrane (7-TM) spanning family of chemokine receptors initially termed "fusin", was identified as the coreceptor for T-tropic HIV-1 variants [10]; the term was changed to C-X-C chemokine receptor type 4 (CXCR4) once its natural ligand was discovered to be stromal derived factor-1 (SDF-1)/CXCL12 [11, 12]. CXCR4 had been previously identified as an orphan receptor called leukocyte-derived seven-transmembrane domain receptor (LESTR), but it only received attention after its isolation as an HIV-1

The knowledge that the β-chemokines macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4 and regulated upon activation normal T-cell expressed and secreted (RANTES)/CCL5 exhibited antiviral activity against M-tropic HIV-1 isolates *in vitro* [14], provided the basis for the discovery of C-C chemokine receptor type 5 (CCR5) as the entry co-factor for these strains by five groups simultaneously [15-19]. The virus thus uses chemokine receptors, mainly CCR5 and CXCR4, to enter susceptible cells, while the cognate chemokine ligands act as natural entry inhibitors. Other chemokine receptors may also function as HIV coreceptors; nonetheless, infection of primary cells via alternative coreceptors is rare and the process can normally be fully blocked by CCR5 or CXCR4 inhibitors [20-22]. Hence, at present, CCR5 and CXCR4 are considered as the only HIV

antiretrovirals targeting ever mutating virus-encoded structures.

perspectives for the clinical use of CCR5 inhibitors will be discussed.

**2. The HIV entry process and the coreceptors** 

**2.1. Discovery of the HIV coreceptors** 

coreceptors of physiological significance.

coreceptor [13].

CCR5 and CXCR4 are two structurally related, heptahelical chemokine receptors that belong to different classes [C-C and CXC, respectively, based on the position of the two conserved cysteine (Cys) residues in their N-termini (Nt)] of the superfamily of G protein-coupled receptors (GPCRs) (reviewed in [13, 23-25]). About half of the drug targets in the pharmaceutical industry are GPCRs, which also, not coincidentally, comprise the largest family of cell-surface receptors [26]. GPCRs become activated by chemokines, small (8-10 kDa) soluble protein ligands that are either promiscuous or specific for a given receptor. Once activated by such extracellular signals, GPCRs undergo conformational changes that trigger the intracellular signal transduction cascade; these series of events begin with the rapid phosphorylation by G protein-coupled receptor kinases (GRKs) predominantly on serine (Ser) and threonine (Thr) residues within the C-tail and third intracellular loop and continue with the activation of heterotrimeric G proteins [27]. In this mode, chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development [28].

CCR5, in particular, appears to play a role in the initiation of adaptive immune responses and the trafficking of effector cells to sites of infection and inflammation as indicated by its expression profile on several effector T cell subsets and antigen-presenting cells, including macrophages, immature dendritic cells and Langerhans cells (reviewed in [22, 29, 30]). Nonetheless, the lack of expression of this gene due to a deletion polymorphism known as "delta32" does not appear to have any deleterious effects; in fact, heterozygosity for the *CCR5*Δ*32* allele is generally associated with delayed disease progression, and homozygosity with incomplete protection from HIV transmission (reviewed in [31]). The observation that its congenital absence does not lead to any overt pathology suggested that CCR5 might be a valid target for pharmacological blockade.

CXCR4, on the other hand, and its single known ligand SDF-1/CXCL12 are both highly conserved [13]; they play an essential role during embryonic development and in several major processes in the adult, including hematopoiesis, leukocyte trafficking in the adaptive immune system, and vascularization [22]. CXCR4 knockout mice show hematopoietic and cardiovascular defects during embryogenesis and die *in utero*; mice lacking SDF-1/CXCL12 are characterized by deficient B-lymphopoiesis and myelopoiesis and abnormal neuronal and cardiovascular development [13]. Clinically tested CXCR4 inhibitors AMD3100, which lacked oral bioavailability, and AMD070 caused significant leukocytosis (reviewed in [22]). The non-cyclam antagonist AMD070 (or AMD11070) is currently in Phase II clinical trials as an HIV cell-entry inhibitor [32]. The development of AMD3100 (by AnorMED) for use in the treatment of HIV infection was terminated, but further studies led to a new indication for this drug; AMD3100 (now Plerixafor, Genzyme) has been approved by the US Food and Drug Administration (FDA) for autologous transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma [32]. The fact that Plerixafor is well tolerated in the developed organism minimizes the concerns of antagonizing such a crucial receptor/ligand axis in mature individuals. CXCR4 antagonists could be used in combination with

antiretrovirals targeting other steps of the viral life cycle; they could also be administered simultaneously with CCR5 antagonists, even as one compound with high affinity for both receptors, as suggested recently [32]. Down-modulating the expression of CXCR4 may certainly be beneficial for some HIV patients [13]. The pharmacologic targeting of CXCR4 may additionally have therapeutic utility for the treatment of acute viral infections of the central nervous system (CNS) [33]. The rest of this chapter focuses on issues related to the development of CCR5 inhibitors.

Chemokine Receptors as Therapeutic Targets in HIV Infection 119

coreceptor [39-42]. Coreceptor engagement of CD4-bound gp120 induces additional reconfigurations, leading to the insertion of the gp41 fusion peptide (FP) into the host cell membrane and the formation of a pre-fusion complex. This pre-fusion intermediate is then refolded into an energetically favorable six-helix bundle that brings the two membranes in close proximity so that fusion can occur; the viral core is thereby released into the cytoplasm

Our understanding of HIV entry has provided invaluable insights into viral tropism and pathogenesis and led to the development of novel classes of antiretroviral agents that inhibit specific stages of the process. CCR5 inhibitors interrupt the viral replication cycle by

Before the identification of the coreceptors, three biological properties were the criteria used to classify the phenotype of HIV variants: (i) the preference for specific target cells (cellular tropism) that distinguished between macrophage (M)-tropic and laboratory T-cell line (T) tropic viruses; (ii) cytopathology, which distinguished between syncytium-inducing (SI) and non-syncytium-inducing (NSI) strains based on the capacity or not to form syncytia (i.e., giant multinucleated cells) through cell fusion in the MT-2 cell line; and (iii) replicative capacity, which mainly considered the *in vitro* growth kinetics of viral strains in culture and distinguished between slow/low (S/L) and rapid/high viruses (R/H) (reviewed in [43]).

The above dichotomies may be explained by the more precise, newly adopted classification system that is based on coreceptor usage; accordingly, viruses that use CCR5 alone or CXCR4 alone for cell entry are currently termed R5 and X4, correspondingly, while variants that can use either coreceptor with comparable efficiency are termed R5X4 [44]. A more appropriate designation for the latter is R5+X4 or D/M (Dual/Mixed), particularly when tropism has not been determined at the clonal level, since many R5X4 isolates are in fact mixtures of R5 plus X4 or, less commonly, either R5 plus dual (R5X4), or even R5 plus dual (R5X4) plus X4, clones [22]. The term R5X4 should thus be reserved for clones that are

**3.2. Determinants of HIV biological phenotype and coreceptor tropism** 

The determinants of coreceptor choice, and thus of biological phenotype, have been mapped to the surface gp120 subunit of Env and primarily to the V3 domain. Swapping the V3 region alone often suffices to switch the coreceptor tropism of a viral clone from R5 to X4 and vice versa [45]. Furthermore, the charge of the V3 region appears to be an important parameter affecting biological phenotype and, tropism, although it alone cannot be used as a marker for phenotype prediction; in general, however, higher net charges (≥ +5) characterize X4 variants compared to the lower V3 region net charges (≤ +4) of R5 variants typically

and a new cycle of infection is initiated.

preventing CCR5 binding and aborting fusion.

**3. Coreceptor use and HIV tropism** 

genuinely capable of using both coreceptors.

**assessment** 

**3.1. Classification systems of HIV biological phenotype** 

#### **2.3. The coreceptors in HIV entry**

Conformationally masked by carbohydrate structures and variable amino acid loops that enable it to evade the humoral immune response, the envelope glycoprotein (Env) on virions is organized into trimers of noncovalently associated surface gp120 and transmembrane gp41 heterodimers (reviewed in [34]). The interaction of HIV with host cell surface receptors trigger the fusogenic potential of Env and allow the virus to enter into its target cells by fusion, presumably at the plasma membrane [35]. The process has long been assumed to occur at the cell surface for such pH-independent viruses as HIV [36], although recent studies support the notion that HIV enters disparate cell types through fusion with endosomes [37, 38]. A schematic of the first steps of the HIV entry process, according to the presently accepted molecular mechanism, is shown in Figure 1.

**Figure 1. Schematic diagram of the first steps of the HIV entry process.** (**A**) HIV entry into target cells is mediated through the interaction of the viral envelope glycoprotein (Env) with host cell surface receptors. (**B**) Surface gp120 first contacts CD4 and then (**C**) a chemokine receptor, typically CCR5 or CXCR4, triggering molecular rearrangements in the gp41 that result in membrane fusion and the initiation of infection.

HIV entry begins with the high affinity binding of gp120 to the host cell CD4, which induces a major conformational change in Env that exposes or creates a binding site on gp120 for the coreceptor, typically either CCR5 or CXCR4. Current structural models suggest that the gp120 "bridging sheet", formed between the constant C1, C2, and C4 domains of gp120 after CD4 binding, interacts with the tyrosine (Tyr)-sulfated coreceptor N-terminus (Nt), while the V3 crown interacts principally with the second extracellular loop (ECL2) region of the coreceptor [39-42]. Coreceptor engagement of CD4-bound gp120 induces additional reconfigurations, leading to the insertion of the gp41 fusion peptide (FP) into the host cell membrane and the formation of a pre-fusion complex. This pre-fusion intermediate is then refolded into an energetically favorable six-helix bundle that brings the two membranes in close proximity so that fusion can occur; the viral core is thereby released into the cytoplasm and a new cycle of infection is initiated.

Our understanding of HIV entry has provided invaluable insights into viral tropism and pathogenesis and led to the development of novel classes of antiretroviral agents that inhibit specific stages of the process. CCR5 inhibitors interrupt the viral replication cycle by preventing CCR5 binding and aborting fusion.
