Chemokines, chemokine receptors and small-molecule antagonists: recent developments

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Chemokines, chemokine receptors and small-molecule antagonists: recent developments James J. Onuffer and Richard Horuk The physiological roles of chemokine receptors have expanded beyond host defense and now represent important targets for intervention in several disease indications. Chemokine receptors have joined the ranks of other members of the G-protein-coupled receptor (GPCR) family in therapeutic potential as small-molecule chemokine receptor antagonists move from discovery to the clinic. Chemokine receptors belong to the rhodopsin family of GPCRs and, as such, are expected to be closely related in structure to other Class A members. In this review, we summarize information that is pertinent to chemokine receptors as therapeutic targets, the status of low molecular weight antagonists in clinical development, molecular modeling of receptor–small-molecule interactions, and the challenges that face drug discovery and development programs. Published online: 10 September 2002

James J. Onuffer* Richard Horuk Dept of Immunology, Berlex Biosciences, Richmond, CA 94806, USA. *e-mail: James_Onuffer@ berlex.com

As a family, G-protein-coupled receptors (GPCRs) have proved to be one of the most fertile protein targets for therapeutic intervention. This class of proteins, with a conserved heptahelical fold, responds to one of the most divergent repertoires of endogenous and exogenous agonist ligands that ranges from light, cations, biogenic amines, pheromones, fragrances and lipids to peptides and globular proteins. Many GPCR-targeted small-molecule drugs have passed clinical trials and are on the market. A more detailed analysis of marketed drugs in this category reveals a strong bias towards biogenic amine receptors such as the 5-HT, histamine, muscarinic acetylcholine and dopamine receptors and adrenoceptors. Thus, the repertoire of new and more effective drugs identified from this category will expand tremendously as small-molecule antagonists of other members of the GPCR family, particularly those of the peptide or protein family, are developed. Chemokine receptors are obvious targets for drug development because they have presumed roles in several disease processes. Most notable of these are functions in acute and chronic inflammation, angiogenesis and angiostasis, and as co-receptors for the cellular entry of HIV (Table 1). The ‘chemotactic cytokine’ or chemokine receptor family is the largest subfamily of peptide-binding GPCRs described thus far (Table 1). Chemokine receptors belong to Class A GPCRs, which are characterized by high homology with rhodopsin, the prototypical family member (Fig. 1a). Also in this family are the biogenic amine receptors that have been successfully targeted by small-molecule antagonists http://tips.trends.com

and thus constitute a solid database of knowledge that should aid the design of novel antagonists for other GPCRs. A wealth of structure–activity relationship (SAR) and mutagenesis studies on the biogenic amine receptors indicate that a generic binding pocket exists within this family. This pocket is made up of residues that line the heptahelical bundle and vary to give each receptor specificity in ligand discrimination [1] (Fig. 1b). With the release of a high-resolution crystal structure of the bovine rhodopsin complex, it is apparent that this generic binding pocket is the structural homolog of the retinal-binding pocket [2–4]. This crystal structure is envisaged to play a major role in the practical development of therapeutics by providing a more relevant template for modeling Class A GPCRs. The modeling process, coupled with high-throughput screening, SAR determination and mapping of contact regions through mutagenesis will rapidly speed the path towards achieving potency and specificity from lead templates. In the past six years, several low molecular weight antagonists have been reported for chemokine receptors. It is not surprising that the compounds obtained share some similarities with molecules identified from biogenic amine drug discovery and development efforts. Data concerning cross-reactivity of these antagonists with other GPCRs and with species selectivity are apparent. Here, we present information about the challenges facing drug development programs that target chemokine receptors. Chemokines and receptors

Initially, in the late 1980s, chemokines and their receptors were described as key players in host defense, based on their potent activity in leukocyte migration and recruitment. Chemokines are classified (CC, CXC, CX3C and XC) based on the number and sequential relationship of the first two of four conserved cysteine residues. Initially, it was envisaged that the CXC chemokine receptors would be important targets for acute inflammation and CC chemokine receptors would be important targets for chronic inflammation. These prototypical chemokines map to human chromosomes 4 (CXC) and 17 (CC) (chromosomes 5 and 11 in mice, respectively). The receptors for these chemokines were also shown to cluster, the prototypical

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a

Table 1. Chemokine receptors and disease association Chemo- Other names kine b receptor

Expression

Ligands

Chromo- Presumed function some

Proposed therapeutic indication

CC CKR1, HM145, MIP-1αR

N, M, MΦ, T, B, Eo, Ba, P, immature DC, mesangial cell

CCL3, 3L1, 5, 7, (9/10), 14, 15, 16, 23

3p21.31

Th1 response

CCR2

CC CKR2B, MCP-1RA, CC CKR2A

M, MΦ, T, Ba, NK, immature DC, endothelium, fibroblast

CCL2, 7, (12), 13, 16

3p21.31

Inflammation

CCR3

CC CKR3, CKR-3, CMKBR3

T (Th2), Eo, Ba, MC, P, DC

CCL5, 7, 8, 11, 13, 3p21.31 15, 24, 26, 28

Th2 response

Multiple sclerosis, rheumatoid arthritis, allograft rejection, viral myocarditis Multiple sclerosis, arthritis, asthma, glomerulonephritis, atherosclerosis Asthma, contact dermatitis

CCR4

CC CKR4, K5-5

T (Th2), Ba, P, immature DC

CCL17, 22

3p24

Th2 response

CCR5

CC CKR5, CMKBR5, ChemR13

M, MΦ, T (Th1), NK, thymocyte, DC, aortic smooth muscle cell

CCL3, 3L1, 4, 5, 8, 14

3p21.31

Th1 response

CCR6

DRY6, CKR-L3, M, MΦ, T, B, immature DC GPR-CY4, STRL22 EB1, BLR2 T, B, DC

CCL20

6q26

Dendritic cell function

CCL19, 21

17q21.1

Lymphocyte and dendritic cell migration to lymph nodes Th2 response

CC CCR1

CCR7

CCR8

Sepsis, asthma, contact dermatitis Multiple sclerosis, rheumatoid arthritis, intestinal inflammation, allograft rejection, HIV infection Psoriasis Cancer

N, M, T, B, thymocyte

CCL1

3p22.1

CCR9

ChemR1, TER-1, GPR-CY6, CKRL1 GPR-9-6

T, thymocyte

CCL25

3p21.31

CCR10

GPR2

T, melanocyte, dermal endothelium, dermal fibroblast, Langerhans cell

CCL27, 28

17q21.2

N, T, M, MΦ, DC, astrocyte, endothelium N, T, M, MΦ, DC, Eo, endothelium

CXCL6, 8

2q35

Neutrophil recruitment

CXCL1, 2, 3, 5, 6, 7, 8

2q35

Neutrophil recruitment, angiogenesis

N, T (Th1), B, DC, Eo, P, mesangial cell, smooth muscle cell

CXCL9, 10, 11

Xq13.1

Th1 response, angiostasis

N, M, MΦ, T, B, DC, P, astrocyte

CXCL12

2q22.1

Organogenesis

Lung reperfusion injury, gout, psoriasis, cancer Lung reperfusion injury, gout, psoriasis, cancer atherosclerosis Multiple sclerosis, rheumatoid arthritis, sarcoidosis, allograft rejection, cancer HIV infection, cancer

M, MΦ, T, B, astrocyte, neuron T, DC

CXCL13 CXCL16

11q23.3 3p21.31

B-cell migration Not known

Not identified Not identified

N, M, MΦ, neuron

CX3CL1

3p21.33

Cell adhesion to endothelia and neurons

T

XCL1, 2

3p21.33

Not known

Allograft rejection, glomerulonephritis, CNS inflammation Not identified

CXC, CX3C and C CXCR1 IL8-RA, Type 1 IL-8R CXCR2 IL-8RB, Type 2 IL-8R CXCR3

GPR9

CXCR4

Fusin, HUMSTR, HM89, LESTR, NPYRL, LCR1 CXCR5 BLR1, MDR15 CXCR6 BONZO, TYMSTR, STRL33 CX3CR1 V28, CMKBRL1, GPR13 XCR1

GPR5

Atopic dermatitis, asthma Intestinal inflammation

Lymphocyte trafficking in thymus and small intestine Lymphocyte trafficking in Skin inflammation, skin and colon ulcerative colitis

a

Abbreviations: B, B cell; Ba, basophil; DC, dendritic cell; Eo, eosinophil; M, monocyte; MC, mast cell; MΦ, macrophage; MIP-1α, macrophage inflammatory protein 1α; N, neutrophil; NK, natural killer cell; P, platelet; T, T cell; Th, T-helper cell. b Chemokine and receptor nomenclature according to [5]. See http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html and http://www.cmbi.kun.nl/7tm/multali/multali.html and [24–28] for detailed information on chemokine ligands and their receptors.

CXC chemokine receptors mapping to human chromosome 2 and the CC receptors mapping to human chromosome 3. These characteristics were unusual and unprecedented in the cytokine field. The apparent redundancy and promiscuity of chemokine interactions http://tips.trends.com

with chemokine receptors has translated to evidence of overlapping but distinct roles in inflammatory sequelae. At present >45 chemokines and almost 20 chemokine receptors have been described [5], and the increasing complexity of this system has broadened

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(a) Class A Rhodopsinlike

461

(b) Class C Class D Class B Calcitonin and Metabotropic Fungal glutamate and pheromone secretin-like pheromone

Class E cAMP receptors (Dictyostelium)

N

Frizzled and smoothenedlike

Protein and peptide

Other

• 5-HT

• Chemokine

• Neuromedin U

• Prostaglandin

• Muscarinic

• Angiotensin

• Opioid

• Prostacyclin

• Purine

• Neuropeptide Y

• fMLP

• Thromboxane

• Adenosine

• Tachykinin

• Thrombin

• Cannabinoid

• Dopamine

• Cholecystokinin • LH

• Histamine

• Endothelin

• TSH

• Octopamine

• Melanocortin

• FSH

• Adrenoceptor • Somatostatin

TM2 TM1

TM5

Rhodopsin Amine

TM3

TM4

TM6

TM7

C

• edg receptors

TM4

TM3 TM2

• Gonadotropin

• Bradykinin

• Orexin and neuropeptide FF

• Bombesin

• Vasopressin and oxytocin

• Neurotensin

• C5a anaphylatoxin

• Galanin

• Proteinase activated

TM5 TM1 TM6

TM7

TRENDS in Pharmacological Sciences

Fig. 1. Chemokine receptors belong to the rhodopsin family of G-protein-coupled receptors (GPCRs). (a) Based on sequence comparison, chemokine receptors are expected to be most similar in structure to Class A (rhodopsin-like) GPCRs. This family includes biogenic amine (monoamine) receptors, which have been targeted successfully by low molecular weight antagonists and agonists. Abbreviations: fMLP, formyl peptide; FSH, follicle stimulating hormone; LH, luteinizing hormone; TSH, thyrotropin. (b) In Class A GPCRs, the hypothetical, generic small-molecule binding site occurs in the heptahelical bundle (indicated by yellow oval, top panel). This site corresponds to the retinal-binding site of rhodopsin. Viewed from the extracellular surface of the receptor (bottom panel), the contacts between the receptor and low molecular weight ligands are shown by arrows. Primary interactions (thick arrows) are observed with contact residues in transmembrane (TM) helices 3, 5, 6 and 7, and minor interactions (thin arrows) from helices 1,2 and 4. Adapted from [1].

the initial oversimplified view of the physiological roles of chemokines and chemokine receptors. Chemokines are a group of small (8–14 kDa), mostly basic heparin-binding proteins that have been shown through crystallographic and nuclear magnetic resonance structure determination to adopt a similar fold, even in cases of low overall sequence identity. Fractalkine is an unusual member of this family because it is synthesized with the structurally conserved chemokine domain at the end of a mucin-rich, transmembrane stalk. The newly discovered CXCR6 ligand CXCL16 has a similar topology. An additional sequence motif (termed ELR) further delineates a subset of the CXC chemokines. The presence of the motif is correlated with angiogenic activity and chemokines that containing ELR are functional ligands for CXCR1 and CXCR2. Historically, the identification and characterization of chemokine-receptor–ligand specificities was based on agonist activity. Recently, it has become clear that the interplay of the receptors and ligands in physiological conditions is complicated by the presence of agonist and antagonist activities [6]. The CXCR3 agonists http://tips.trends.com

I-TAC (interferon-inducible T-cell α chemoattractant; CXCL11), MIG (monokine-induced by γ-interferon; CXCL9) and IP10 (interferon-inducible protein 10; CXCL10) involved in responses driven by T helper 1 (Th1) cells have been reported to be antagonists of CCR3, which is postulated to play a role in Th2-driven responses [7]. Additionally, the CCR3 agonist eotaxin (CCL11) is a partial agonist for CCR2 [8] and a weak agonist for CCR5 [9], whereas the CCR1/CCR2/CCR3 agonist MCP-3 (monocyte chemotactic protein 3; CCL7) is an antagonist for CCR5 [10]. A thorough characterization of the binding of all known chemokine ligands with all known receptors is expected to reveal additional examples of cross-reactivity between chemokine ligands and receptors. Structure–function studies indicate that chemokines have two major sites of interaction with their cognate receptors, comprising the flexible N-terminal portion that precedes the first cysteine and the rigid loop that follows the second cysteine. The relative importance of each of these two contact regions to overall ligand affinity varies depending on the receptor examined and reflects synergy between several important contacts [11]. A similar synergy of contact regions exists on the receptor end with regard to N-terminal sequence and extracellular loop dependence of chemokine binding. The flexible N-terminal sequence of chemokines plays an important role in determining agonist activity [6]. N-terminal modification of RANTES (regulated on activation, normal T-cell expressed and secreted; CCL5) by addition of a methionine results in an antagonist [12]. N-terminal modification of RANTES by addition of an

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Table 2. Small-molecule chemokine receptor antagonists reported to be in clinical development

a,b

Receptor

Compound

Company

Clinical phase

Delivery

Indication

CCR1

BX471 (ZK811752)

Berlex

Phase I

Oral

Not AZD8309, disclosed AZD7140

AstraZeneca

Early clinical phase –

CCR3

DPC168

Bristol-Myers Squibb

Phase I

CCR5

CCR5

SCHC Schering-Plough Phase I Oral (Sch351125) (study terminated) SCHC Schering-Plough Phase I Oral (Sch351125) UK427857 Pfizer Phase I –

Autoimmune disease (multiple sclerosis) Rheumatoid Structures and delivery route unavailable for arthritis review; receptor target is not specifically identified (macrophage inflammatory protein 1α antagonists) Allergic disease Structure and delivery route unavailable for (asthma, allergic review rhinitis, eczema) HIV Study terminated in part because QTc prolongation observed at highest doses HIV Initiated in December 2001 in France

CXCR4

AMD3100

AnorMED

Phase Ia/IIb (development terminated)

Intravenous

CXCR4

AMD3100

AnorMED

Phase I

Subcutaneous Mobilization of injection white blood cells

CCR5

–

HIV HIV

Notes

Structure and delivery route unavailable for review Development terminated for HIV because premature ventricular beats observed in high-dose cohorts and inability to reach dose criteria for efficacy −

a

Information current as of April 2002 and compiled from the Investigational Drug Database (Iddb3; http://www.iddb3.com). This information is used with the permission of Current Drugs Limited. (Copyright 2002 Current Drugs Limited. All rights reserved.) b Available structures are shown in Fig. 2.

aminooxypentane group results in a chemokine that is an agonist for CCR3 but a partial agonist for CCR5 and, to a lesser extent, CCR1 [13,14]. The complexity of chemokine function is further exemplified by reports of N-terminal proteolytic processing of selected chemokines by CD26, attractin, plasmin and urokinase plasminogen activator that is reminiscent of the processing that occurs with some neuropeptides [15–20]. This processing of chemokines affects not only agonist activity but also receptor specificity. Additionally, the transmembrane protease ADAM17 (TACE; tumor necrosis factor α convertase) has been shown to be responsible for the PMA (phorbol 12-myristate 13-acetate)-induced cleavage and shedding of fractalkine from the cell surface [21]. This cleavage converts the membrane-bound adhesion molecule to a soluble chemoattractant. Recently, it has been reported that mouse fractalkine (CX3CL1) and TARC (thymus and activation-regulated chemokine; CCL17), which are tightly linked on mouse chromosome 8, share a signal sequence in several tissues [22]. Thus, the tissue specificity of TARC expression in mice is regulated by two independent mechanisms, one by transcription from the TARC promoter and the second from the promoter of fractalkine followed by tissue-specific alternative splicing. Similarly, the human chemokines HCC-1 (hemofiltrate CC chemokine; CCL14a), HCC-3 (CCL14b) and HCC-2 (CCL15) are encoded by monocistronic and bicistronic transcripts that are alternatively spliced [23]. Whether a similar situation occurs within other clusters of chemokine genes remains to be demonstrated. The development of a detailed interaction web of agonist and antagonist http://tips.trends.com

activities combined with expression levels in pathological conditions is expected to reveal additional fine-tuning of chemokine-dependent responses in vivo. Assigning potential targets of disease states

Because of the inherent complexity of the chemokine–chemokine-receptor axis, determined by expression patterns, post-translational modifications and the overlapping ligand–receptor specificities, the primary indication of therapeutic potential for this family comes from examining results from knockout animals in models of human disease states. However, because chemokines and chemokine receptors have roles in hematopoiesis and development, responses in knockout animals might not reflect the effect of therapeutic intervention accurately. Ultimately, examination of the effects of low molecular weight antagonists in vivo will provide the additional evidence required for target validation. Several potent and selective antagonists have been identified for the major chemokine receptors that are targeted by current drug development programs (CCR1, CCR2, CCR3, CCR5, CXCR2, CXCR3 and CXCR4). Detailed information on these and other chemokine receptors, and the antagonists that target them can be found in several recent reviews [24–28]. Of the many reported chemokine receptor antagonists, relatively few have progressed to clinical trial (Table 2). The results from trials with these inhibitors will help set the stage for future compound development. General features of chemokine receptor antagonists

Chemokine receptor antagonists appear to share common features. First, the majority of these molecules

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H3C O O O Cl

N

CH3 F

N N

NH

Br

N

NH HN

O−

CH3 H3C

NH

N+

N

N O

NH2

HN

NH HN O

BX471(Berlex) (CCR1 antagonist)

N

CH3

SCHC (Schering-Plough) (CCR5 antagonist)

AMD3100 (AnorMED) (CXCR4 antagonist)

O H N

O

O N N

H3C

NH

O

CH3 CH3

O

E913 (Ono) (CCR5 antagonist)

NH HN

N

TAK779 (Takeda) (CCR5 antagonist)

F

NH HN

N

F CH3

AMD3389 (AnorMED) (CXCR4 antagonist)

O O

N+

O

NH

H N

Cl−

NH HN

F

RS102895 (Roche) (CCR2 Antagonist)

Cyclam (AnorMED) (CXCR4 antagonist) TRENDS in Pharmacological Sciences

Fig. 2. Structures of chemokine receptor antagonists mentioned in this review.

contain a basic region, exemplified by the presence of piperidine, piperazine, spiropiperidine, pyrrolidine, guanidine, quaternary nitrogen or bicyclam groups. Second, many of them appear to have a preference for halogen-modified aromatic rings. The molecular basis for potent antagonism of CCR2 by the Roche spiropiperidine-derived compounds appears to involve an ionic interaction of the basic nitrogen of the spiropiperidine with an acidic glutamate at position 291 [29]. This glutamate residue is conserved in the majority of chemokine receptors and indicates a common mode of interaction within this family. Piperidine-containing compounds represent one of the most common templates identified for GPCRs, as indicated by the presence of this group in several small-molecule compounds that are either potent antagonists or agonists of several receptors, including those for chemokines, somatostatin, C5a, tachykinin, neuropeptide Y and cholecystokinin [1]. Although the similarity of these structures could result from the use of similarly derived chemical libraries, it also indicates the existence of a conserved binding pocket within the transmembrane region of GPCRs. The nature and location of this common binding pocket has been described recently and is structurally analogous to the binding pocket occupied by retinal in the rhodopsin crystal structure. Several important residues in biogenic amine receptors that interact with low molecular weight ligands map to the corresponding retinal-binding site in rhodopsin [2,3]. http://tips.trends.com

Limited antagonist-mapping studies through SAR and mutagenesis have been reported for chemokine receptors. In addition to the mapping of RS102895 (Fig. 2), a spiropiperidine-containing compound, to CCR2 [29], mapping of CCR5 residues that interact with TAK779 (Fig. 2) [30] and CXCR4 residues that interact with AMD3100 (Fig. 2), a bicyclam antagonist of CXCR4 [31–33], have been reported. The results of these studies, referenced to the rhodopsin crystal structure, are indicated in Fig. 3. Such analysis reveals that the binding-site residues identified for these antagonists appear to map to the immediate vicinity of the structurally conserved retinal-binding site of rhodopsin. Because chemokines are relatively large ligands compared with biogenic amines, it is apparent that there is a potential discrepancy with regard to the nature of antagonism by low molecular weight ligands for these two GPCR subclasses. Antagonists of biogenic amine receptors are expected to be competitive because the agonist- and antagonist-binding sites are shared in most cases (a simplistic generalization). However, antagonism of chemokine receptors is generally presumed to be indirect and involve antagonist-induced structural changes in the receptor that preclude chemokine binding. Given this, it might be possible to identify chemokine receptor antagonists that inhibit function without displacing chemokine binding. To date, only one chemokine receptor antagonist that partially exhibits this property has been reported [34], which

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(a)

Helix 1

Rhodopsin Human CCR2 Human CCR5 Human CXCR4

MNGTEGPNFYVP--FSNKTGVVRSPFEAPQYY-------------LAEPWQFSMLAAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLN MLSTSRSRFIRNTNESGEEVTTFFDYDY----GAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTD MDYQVSSP-IYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILINCKRLKSMTD MEG-----ISIYT-SDNYT-EEMGSG-DYD---SMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTD

Rhodopsin Human CCR2 Human CCR5 Human CXCR4

YILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGE-NHAIMGVAFTWVMA IYLLNLAISDLLFLITLPLWAHSAANE--WVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVA IYLLNLAISDLFFLLTVPFWAHYAAAQ--WDFGNTMCQLLTGLYFIGFFSGIFFIILLTIDRYLAVVHAVFALKARTVTFGVVTSVITWVVA KYRLHLSVADLLFVITLPFWAVDAVAN--WYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPA

Rhodopsin Human CCR2 Human CCR5 Human CXCR4

LACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETN--NESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMV VFASVPGII-FTKCQKEDSVYVCG-----PYFP-RGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCR-NEKK-------RHRAVRVI VFASLPGII-FTRSQKEGLHYTCSSHF--PYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCR-NEKK-------RHRAVRLI LLLTIPDFI-FANVSEADDRYICDR-FY-PND---LWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQ---------KRKALKTT

Rhodopsin Human CCR2 Human CCR5 Human CXCR4

IIMVIAFLICWLPYAGVAFYIFTHQG---------SDFGPIFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCCG-KNPLGDDEASTT FTIMIVYFLFWTPYNIVILLNTFQEFFGLSN-CESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFY FTIMIVYFLFWAPYNIVLLLNTFQEFFGLNN-CSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQ VILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGK

Rhodopsin Human CCR2 Human CCR5 Human CXCR4

VSKTE--TSQVAPA RETVDGVTSTNTPSTGEQEVSAGL QEAPERASSVYTRSTGEQEISVGL RGGHSSVSTESESSSFHSS

Helix 2

Helix 3

Helix 4

Helix 5

Helix 6

Helix 7

Residues within 7Å of retinal in bovine rhodopsin structure Residues mapped by mutagenesis on chemokine receptors

(b) Bovine rhodopsin

C

P

Extracellular

C L

P

G N L

A W

D L

Y W P

L

C Y

P

N P Y

R Y F

Cytoplasmic

Residues within 7Å of retinal in bovine rhodopsin structure Residues mapped by mutagenesis on chemokine receptors

TRENDS in Pharmacological Sciences

Fig. 3. Comparison of the amino acid residues of chemokine receptors that interact with low molecular weight antagonists with the retinal-binding site of rhodopsin. (a) Sequence alignment of human chemokine receptors CCR2, CCR5 and CXCR4 with bovine rhodopsin. Conserved residues are indicated in bold. Transmembrane helices, determined from the crystal structure of rhodopsin, are indicated by shaded boxes [4]. Rhodopsin residues that are within 7 Å of the bound retinal and the corresponding residues of CCR2, CCR5 and CXCR4 are highlighted in cyan. Limited mutagenesis studies of chemokine receptors show that residues highlighted in red are important for binding low molecular weight antagonists [29–33]. There is significant overlap between several transmembrane helices and the extracellular loop 2 (ECL2) between helices 4 and 5. (b) Mapping information from chemokine antagonists in the context of a two-dimensional model of rhodopsin, based on the alignment in (a).

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could indicate that productive antagonist binding and structural changes are tightly coupled. Alternatively, this might result from the fact that most inhibitor-screening programs use chemokine binding as the primary screen. Additional information can also be derived from the literature regarding the nature of the antagonist-binding site and the structural changes that result from antagonist binding to chemokine receptors by the use of epitope-specific monoclonal antibodies. The lid of the binding pocket of retinal is covered by extracellular loop 2 (ECL2), which contains

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(a)

3 4 2

5 1 7

6

(b)

TM4

TM3 C C

TM2

TM5 TM1 TM6 CCR5 Extracellular loop 2 (ECL2)

ECL2a

TM7 ECL2b

FTRSQKEGLHYTCSSHFPYSQYQ mAb 2D7 (ECL2a)

Antibody binding in presence of antagonist TAK779 (Takeda), E913 (Ono)

+

mAb 45531 (ECL2b) –

TRENDS in Pharmacological Sciences

Fig. 4. The location and structural features of extracellular loop 2 (ECL2) between transmembrane helices 4 and 5 of rhodopsin is expected to be similar to that of chemokine receptors. (a) The location of ECL2 in the crystal structure of rhodopsin [4] is shown from an extracellular viewpoint with an N-terminal portion of the receptor removed for clarity. Transmembrane helices (green) are numbered. Bound retinal is shown in yellow. ECL2 contains two antiparallel β-strands (pink and blue) that line the top of the retinal pocket. The second β-strand (blue) is proximal to the bound retinal. (b) Recent experiments indicate that the recognition of several epitopes of ECL2 in chemokine receptors is influenced by the presence of low molecular weight antagonists [30,31,35]. ECL2 of chemokine receptors can be further delineated into ECL2a and ECL2b regions, based on the position of a conserved cysteine that forms a disulfide bond with a cysteine near the extracellular portion of transmembrane helix 3 (a feature shared with rhodopsin). This conserved cysteine is located in the second β-strand (blue) of the loop. The β-strand in ECL2a is indicated in pink and the antagonist-binding pocket, predicted by homology to rhodopsin, is indicated in yellow. The anti-chemokine receptor CCR5 monoclonal antibody(mAb) 2D7, which recognizes epitopes of ECL2a, is insensitive to the binding of the structurally dissimilar CCR5 antagonists TAK779 and E913. However, the anti-CCR5 mAb 45531, which recognizes epitopes of ECL2b, is antagonized by both TAK779 and E913, which indicates that low molecular weight antagonists influence the structure and/or accessibility of this portion of the loop. Similar modulation of monoclonal-antibody recognition of ECL2 by antagonists has been reported for CXCR4.

a conserved, antiparallel β-strand motif (Fig. 4a). This loop contains a conserved cysteine residue that anchors ECL2 to a conserved cysteine residue located near the extracellular surface of helix 3. In the rhodopsin structure the second portion of this loop (ECL2b) is proximal to the bound retinal, whereas the first portion http://tips.trends.com

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of the loop (ECL2a) lies on top of the second strand and consequently more of its surface is exposed. The structurally dissimilar CCR5 antagonists TAK779 and E913 (Fig. 2) inhibit the binding of monoclonal antibodies directed at ECL2b but have no effect on antibody recognition by ECL2a antibodies [30,35] (Fig. 4b). Similarly, AMD3100 inhibits the binding of the neutralizing monoclonal antibody 12G5, which binds to several surface epitopes on CXCR4, including ECL2 [31]. Surprisingly, the monocyclam AMD3389 (Fig. 2) enhances the binding of 12G5 to CXCR4, although this feature is not observed for the cyclam derivative of AMD3100 (Fig. 2). These results support a potential mechanism for antagonist effects on chemokine binding and subsequent function through modulation of the structure of the extracellular loops of the receptor. The importance of the ECL2 loop in determining chemokine binding and selectivity has been established previously for CCR5 [36]. Molecular models of chemokine receptors complexed with chemokines and low molecular weight antagonists have been published recently. A model of CCR2 interacting with RS102895 has been reported [29]. A structural model of CXCR4 interacting with AMD3100 has been proposed [31]. Three models of CCR5 have been reported. One models the interaction between CCR5 and TAK779 [30] and the other two include the additional modeling of interactions between CD4 and GP120 [37,38]. The validity and refinement of these and future models rely extensively on mutagenesis and SAR data. Additional information to refine these models will come from comparative SAR of series of antagonist compounds on related receptors from other species. The differences in the cross-reactivity of antagonists at CCR1, CCR2 and CCR3 with the corresponding mouse receptors provide the groundwork for such efforts [39–41]. Challenges facing inhibitor development programs

Because chemokine receptors are closely related to other Class A GPCR members it is not surprising to find examples of small-molecule antagonists that cross-react with other GPCRs. This appears to be particularly true for several chemokine receptor antagonists, which cross-react with biogenic amine receptors. For example, Schering-Plough’s piperazine-based CCR5 inhibitors cross-react with muscarinic acetylcholine receptors [42], whereas Roche’s CCR2 spiropiperidine inhibitor series cross-reacts with 5-HT1A receptors in addition to α1-adrenoceptors [29]. Furthermore, one of SmithKline Beecham’s CCR2 inhibitors cross-reacts with 5-HT receptors [43] and Berlex’s 4-hydroxypiperidine series of CCR1 inhibitors cross-reacts with several biogenic amine receptors, including dopamine and muscarinic receptors [44]. Merck’s CCR5 inhibitors are derived from templates identified in their tachykinin-receptor-antagonist program. Surprisingly, as shown by these examples, the selectivity of chemokine receptor antagonists for members of the biogenic amine subclass of receptors

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Acknowledgements We would like to acknowledge researchers whose work is not specifically referenced owing to space limitations.

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(adrenoceptors, and muscarinic, 5-HT and dopamine receptors) appears to present more of a potential problem than other peptide ligand GPCRs (including other chemokine receptors). Because selectivity issues can limit the potential therapeutic use of these receptors, a more detailed understanding of the binding-site interactions is necessary to ‘dial out’ undesired reactivities while maintaining potency at the chemokine receptor target. Another problem facing this field is the lack of relevant animal models. This problem is manifested in several ways. First, there are species differences in the expression of several chemokines and receptors. For example, interleukin 8 does not exist in mice. Mice also do not have CXCR1, although they have a CXCR2 homolog. Humans appear to have a more diverse array of chemokine ligands compared with mice. As an example, two macrophage inflammatory protein 1α (MIP-1α) genes can be present in humans. These genes encode MIP-1α variants CCL3 and CCL3L1, which have different regulatory pathways and receptor potencies. Second, the expression patterns of chemokines and chemokine receptors is often different in rodents. For example, CCR1 is highly expressed in neutrophils in mice, which leads to a strong neutrophil component in CCR1-dependent inflammation. The concentration of CCR1 is much lower in human neutrophils, although this can be altered by culture conditions [45]. Species-dependent expression differences have also been noted for CX3CR1 [46] and CCR5 [47]. Third, there are differences in the selectivity of chemokines for chemokine receptors in humans compared with mice. Mouse RANTES and MCP-3 are reported to lack affinity for mouse CCR1 but are functional ligands for the human CCR1 receptor [48]. Lastly, there are known problems with species selectivities of several small-molecule GPCR inhibitors as exemplified by tachykinin receptor antagonists [49]. CCR1 antagonists from Berlex and CCR1/CCR3 antagonists from Banyu discriminate between human and mouse receptors [39,40]. Additionally, it is reported that the Roche CCR2 spiropiperidine series is selective for primate receptors because it has negligible cross-reactivity with mouse and rat CCR2 receptors [41]. Further examples are expected as the results of additional drug development programs targeted at chemokine receptors are revealed. One approach to dealing with this issue is to create ‘knock-in’ animals in which the murine receptor is replaced by the human form. Alternatively, development strategies might use primate models. A third potential hurdle facing development programs is the nature of the screening assay employed. Conformational heterogeneity has been reported for chemokine receptors. CCR5 receptors exist in multiple conformations, populations of which can be identified using monoclonal antibodies [50,51]. A similar situation has been reported for the CXCR4 receptor [52]. Gupta et al. demonstrated that, in cell lines, binding of SDF-1 (stromal cell-derived factor 1; CXCL12) to the http://tips.trends.com

CXCR4 receptor is displaced biphasically by AMD3100, whereas the inhibition of SDF-1 functional responses is monophasic [53]. Conformational heterogeneity of CCR5 and CXCR4 has been discussed recently with regard to the variability in the use of these receptors by HIV [54]. Do these results indicate that a functional screen is more reliable than the more commonly employed binding assay? What occurs when cell membranes rather than whole cells are used? The binding of several chemokines to the CXCR3 receptor depends on the G-protein-coupling state of the receptor [55]. In this study, the chemokines CXCL10 and CXCL11 acted as noncompetitive or allotopic agonists for CXCR3 that exhibit differential binding to different activation states of the receptor. Similarly, binding of MIP-1β (CCL4) by CCR5 is dependent on G-protein coupling [56]. These results suggest the possibility that cells might present different chemokine receptor conformations or activation states that might exhibit differential coupling to selected chemokine ligands. The conformation and behavior of the chemokine receptor target might depend on the cell line used, particularly because different cell lines have distinct complements of G-protein subunits, surface receptors and membrane lipids, each of which might influence the behavior of chemokine receptors. For example, CCR5 physically associates with CD4 [57], CXCR4 physically associates with CD26 [58] and CCR2 activity is influenced by the presence of glycosylphosphatidylinositol (GPI)-linked proteins on the cell surface [59]. Should relevant small-molecule screens be performed in the presence of these proteins? Will antagonists identified from such screens be potent antagonists in vivo? These and other questions will be answered in due course as small-molecule antagonists progress through Phase II and Phase III clinical trials. For the present, it is vitally important to verify similar behavior and potencies of small-molecule leads on primary cell systems to validate the screening system employed. Results from functional assays will have the highest relevance to therapeutic intervention. Concluding remarks

In the late 1980s scientists isolated the signaling molecules, termed chemokines, that allowed leukocytes to communicate with one another and seek out and destroy invading pathogens. However, the immune response is a double-edged sword and can, under some circumstances, be activated inappropriately and targeted towards normal, healthy tissue, which leads to autoimmunity and disease. In the space of 6 years, numerous, non-peptide chemokine receptor antagonists have been identified and some have advanced to clinical trials. As knowledge of the role of chemokine receptors has expanded from autoimmunity to AIDS, the importance of these intervention therapies has grown. The promise of highly specific therapies for several diseases, based on chemokine receptor antagonists, is on the horizon.

Review

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