Chemokine Receptors: Overview

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Chemokine Receptors: Overview Philip M. Murphy* Molecular Signaling Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA * corresponding author tel: 301-496-2877, fax: 301-402-4369, e-mail: [email protected] DOI: 10.1006/rwcy.2000.02012.

INTRODUCTION Chemokine receptors are defined by their ability to bind chemokines in a specific and saturable manner, and to transduce a cellular response. At the molecular level, this definition has been met by 16 human cell surface proteins (named CXCR1 to CXCR5, CCR1 to CCR9, XCR1, and CX3CR1), which together comprise the largest known structurally defined division of the rhodopsin superfamily of seven transmembrane domain, G protein-coupled receptors (GPCRs) (Murphy, 1994; Premack and Schall, 1996; Yoshie et al., 1997; Locati and Murphy, 1999; Zlotnik et al., 1999). In addition, four herpesvirus-encoded chemokine receptors (ORFs US28 of human cytomegalovirus, ECRF3 of herpesvirus saimiri, UL12 of HHV-6 and no. 74 of HHV-8/Kaposi's sarcoma herpesvirus (KSHV; also known as KSHV GPCR) (Isegawa et al., 1998; Pease and Murphy, 1998), and two nonsignaling mammalian chemokine-binding proteins (D6 and the Duffy antigen receptor for chemokines, DARC) (Horuk et al., 1994; Nibbs et al., 1997) have been described. Studied initially for their roles in leukocyte trafficking, chemokine receptors are now known to have multiple additional functions, including regulation of development of the cardiovascular, gastrointestinal, immune, and central nervous systems (Tachibana et al., 1998; Zou et al., 1998), and usage as cell entry factors by HIV-1 (Cocchi et al., 1995; Feng et al., 1996; Berger et al., 1999) and Plasmodium vivax (Horuk et al., 1993), the causative agents of AIDS and a form of malaria, respectively. The purpose of this chapter is to provide an overview of the shared and differential features of these molecules, as

introduction to the chapters devoted to individual chemokine receptors.

CHEMOKINE RECEPTOR STRUCTURE The deduced amino acid sequences of chemokine receptors have 25±80% identity, indicating a common ancestry. This has facilitated discovery of additional family members by crosshybridization of cDNAs and genes to DNA probes from known receptors (Murphy, 1996). The structural boundary which separates chemokine receptors as a group from other types of G protein-coupled receptors is not sharp, and they lack a single structural signature. However, several structural fea-tures common to known chemokine receptors are less common in other types of G protein-coupled receptors, including a length of 340±370 amino acids; a highly acidic N- terminal segment; the sequence DRYLAIVHA, or a minor variation thereof, in the second intracellular loop; a short basic third intracellular loop, and a cysteine in each of the four extracellular domains (Murphy, 1994). A novel sequence which contains all of these features is likely to represent a chemokine receptor. The folded structure of chemokine receptors has not been determined; however a model can be constructed for the transmembrane helices based on the known structure of rhodopsin (Unger et al., 1997). In addition, domain-specific antibodies have been used in some cases to establish the transmembrane topography of N- and C-termini and the loop regions, which are consistent with the rhodopsin model. Early biochemical

1972 Philip M. Murphy crosslinking data were consistent with a monomer structure for neutrophil IL-8 receptors (Moser et al., 1991) and Duffy (Neote et al., 1993), whereas more recent data consistent with a dimer have been reported for CCR2 (Rodriguez-Frade et al., 1999), CCR5 (Benkirane et al., 1997), and CXCR4 (Lapham et al., 1999). In the case of CCR2, a dimer has been implicated as the functional form of the receptor. This has further stimulated the unsettled debate as to whether chemokines bind to receptors as monomers or dimers. Although they exist as monomers at physiologic concentrations, most chemokines form dimers at high concentrations. A major unanswered question is how chemokines bind to receptors. Mutagenesis studies support a Velcro-like interaction between ligand and receptor, in which multiple low-affinity binding sites integrate to produce an overall high-affinity binding energy (Ahuja et al., 1996; Berson and Doms, 1998; Paavola et al., 1998). There appear to be two classes of binding sites: the first for chemokine docking to receptor, the second for receptor triggering. Because of the seven transmembrane domains, the binding sites are not contiguous in the primary sequence, but instead are gathered together

dirndl-like in the folded protein. The chemokine Nterminus is not usually important for docking but is typically critical for activation. Putative roles of the transmembrane domains include determination of the conformation of the extracellular domains, contact with the chemokine activation domain, and signal transduction. Additional functional domains include the C-terminus, which typically contains multiple serine and threonine residues, some of which may be phosphorylated upon activation and may act to desensitize the receptor (Ali et al., 1999), domains in the second and third intracellular loops important for G proteinbinding and activation (Damaj et al., 1996), and a conserved consensus sequence for tyrosine sulfation in the N-terminus (Farzan et al., 1999), which in the case of CCR5 must be utilized for HIV coreceptor activity.

CHEMOKINE RECEPTOR SPECIFICITY Chemokine receptors can bind multiple chemokines, and vice versa; however the specificities are generally restricted by chemokine class (Table 1 and Table 2).

Table 1 Chemokine receptor classification and specificity Class

Subtype

Ligands

CC

CCR1

MIP-1, RANTES, MCP-3

CCR2

a

CCR3

a

MCP-1, MCP-3, MCP-4 Eotaxin, eotaxin 2, RANTES, MCP-2, MCP-3, MCP-4, MCP-5

CCR4

TARC, MDC

CCR5a

MIP-1, MIP-1 , RANTES, MCP-2

CCR6

LARC

CCR7 CCR8

ELC, SLC a

I-309

CCR9

TECK a

CXC

CC/CXC

CMV US28

MIP-1, MIP-1 , RANTES, MCP-1, fractalkine

HHV-6 UL12

MIP-1, MIP-1 , RANTES, MCP-1

CXCR1

IL-8, GCP-2

CXCR2

IL-8, GRO, GRO , GRO , NAP-2, ENA-78, GCP-2

CXCR3

MIG, IP-10, I-TAC, eotaxin, SLC

CXCR4a

SDF-1

ECRF3

IL-8, GRO, NAP-2

KSHV GPCR

IL-8, GRO, RANTES, MCP-1, I-309

C

XCR1

Lymphotactin

CX3C

CX3CR1a

Fractalkine

Italicized receptors are viral in origin. All others are human. a HIV-1 coreceptors.

Chemokine Receptors: Overview 1973 Table 2 Chemokine-binding proteins with seven transmembrane domain motif, not yet demonstrated to signal Molecule

Ligands

Duffy

IL-8, NAP-2, GRO, I-309, RANTES, MCP-1

D6

MIP-1, RANTES, MCP-1

This provides for a simple nomenclature in which each receptor is named by the chemokine class it recognizes, followed by the letter R and an arabic numeral assigned by the order of discovery relative to other receptors. Thus, CCR1 is the first receptor specific for CC chemokines to be discovered. There are several known exceptions to this rule: CXCR3 also binds two CC chemokines, eotaxin and SLC, but apparently with lower affinity than its CXC ligands IP-10, MIG, and I-TAC (Soto et al., 1998; Weng et al., 1998); KSHV GPCR binds multiple CC and CXC chemokines with comparable affinity (Arvanitakis et al., 1997); US28 binds multiple CC chemokines and the CX3C chemokine fractalkine (Kledal et al., 1998); and Duffy binds multiple CC and CXC chemokines with comparable high affinity (Chaudhuri et al., 1994). Each receptor has a distinct specificity for chemokine ligands and leukocyte subsets; however, the specificities may overlap considerably. For example, CCR1 and CCR5 both bind MIP-1 and RANTES but can be distinguished by their specificities for MCP-3 and MIP-1 respectively. The structural basis of specificity is counterintuitive. For example, CCR2 is much more related structurally to CCR5 than is CCR1 (82% versus 56% amino acid identity), but has only one high-affinity ligand in common with CCR5, whereas CCR1 has several. Instead, CCR2 shares several ligands with CCR1, yet the two receptors are only 56% identical in amino acid sequence. Promiscuous chemokine ligand±receptor relationships are common. As a result, defining the chemokine receptor responsible for stimulus±response coupling in primary cells is often not straightforward due to overlapping specificities of receptors for ligands and leukocytes, and a paucity of receptor subtype-selective blocking agents. Not only can distinct receptor subtypes specific for the same chemokine and the same function be coexpressed on the same cell, but also distinct chemokines acting at separate receptors coexpressed on the same cell can induce the same cellular response. Although antireceptor monoclonal antibodies and mice with targeted gene disruptions are now being used to resolve specificities in vivo, problems of interpretation persist

due to the inequality of chemokine and chemokine receptor repertoires, tissue distribution, and biological usage among species. For example, IL-8 is found in human and rabbit, but not in mouse, and CXCR1 is expressed mainly in human neutrophils versus rat macrophages (Dunstan et al., 1996). Fortunately, this situation appears to affect only a minority of chemokine receptors. Chemokine receptors have been described on all leukocyte subsets studied (Luster, 1998; Mantovani et al., 1998). Details of expression and regulation are complex, and in some cases controversial, and are beyond the scope of this overview; however, a few key patterns can be summarized. Cells of the granulocyte series appear to express a limited repertoire of chemokine receptors. Neutrophils express predominantly CXCR1 and CXCR2, whereas human eosinophils express mainly CCR3, the IL-8 receptors and, to a more variable extent, CCR1. CCR3 has also been reported on basophils and a small subset of TH2 T lymphocytes, consistent with a role in allergic inflammation. Monocytes and macrophages express a broader repertoire, which includes CXCR1, CXCR2, CXCR4, CCR1, CCR2, CCR5, CCR8, and CX3CR1. T lymphocytes express the complete chemokine receptor repertoire, but in an incomplete and differential manner on specific subsets. TH1 and TH2 cells have distinct receptor repertoires. Of note, CXCR3 and CCR4 have been reported to be markers of TH1 and TH2 cells, respectively, although this has been challenged. Little information has been reported for mature B cells, although it is clear that CXCR5 is expressed at high levels and is functionally important. Additional information on this subject with extensive lists of primary references can be found in the individual receptor chapters.

CHEMOKINE RECEPTOR SIGNALING Aspects of signaling common to all known mammalian chemokine receptors include induction of calcium flux and chemotaxis, and marked inhibition of both

1974 Philip M. Murphy by Bordetella pertussis toxin (Bokoch, 1995; Ward et al., 1998). The latter reflects coupling of receptors in primary cells to Gi-type heterotrimeric G proteins, whose  subunits are covalently ADP-ribosylated by pertussis toxin, which inactivates the protein. There are several noteworthy exceptions to this. First, constitutive signaling by the viral receptor KSHV GPCR is completely insensitive to pertussis toxin; the presumptive G protein involved has not yet been identified (Arvanitakis et al., 1997). Second, although CX3CR1 can signal in a conventional pertussis toxinsensitive chemotactic pathway in response to its soluble chemokine ligand fractalkine, it can also function as a powerful cell±cell adhesion molecule by binding to membrane-bound fractalkine in a pertussis toxin-insensitive manner (Imai et al., 1997). Third, inhibition of chemokine action in primary cells by pertussis toxin is often incomplete, perhaps reflecting coupling to other classes of G proteins. Consistent with this, several chemokine receptors, including CCR1, CXCR1, CXCR2, and CCR2, signal in a pertussis toxin-insensitive manner in cell lines cotransfected to express receptor and proteins from the Gq class, including G16 which is preferentially expressed in hematopoietic cells (Kuang et al., 1996; Xie et al., 1997). Signaling by the IL-8 receptors, CXCR1 and CXCR2, has been studied most extensively, and includes additional common elements such as stimulation of phospholipase C 2 and inhibition of adenylyl cyclase (Hall et al., 1999), and at least two differences: selective activation of phospholipase D and the NADPH oxidase by CXCR1 (Jones et al., 1996). Activation of CCR1 and CCR2 has also been shown to inhibit adenylyl cyclase (Myers et al., 1995), consistent with coupling to Gi. Activation of several chemokine receptors has been associated with protein tyrosine phosphorylation, including pyk-2 in the case of CCR5 (Davis et al., 1997). IL-8 signaling to the Ras/Raf/MAP kinase/PI-3 kinase pathway has also been reported (Knall et al., 1996). Unusual dual signaling pathways have been reported for RANTES in T cells. At low concentrations pertussis toxinsensitive calcium flux occurs, whereas at high concentrations, threshold  1 mM, activation of ZAP70 in a pertussis toxin-insensitive manner is observed. The receptor mechanism for this latter phenomenon has not yet been defined (Bacon et al., 1995). Activation of chemokine receptors ultimately results in desensitization, which has been associated with phosphorylation of serines and threonines in the C tail, and clathrin-mediated endocytosis (Ali et al., 1999; Oppermann et al., 1999; Yang et al., 1999). This process may be important for receptor resensitization and chemotaxis. Signaling is not required for HIV

coreceptor activity by CCR5 or CXCR4 (Alkhatib et al., 1997; Amara et al., 1997; Farzan et al., 1997), or its inhibition by cognate chemokines, or, as mentioned previously, for proadhesive activity by CX3CR1 (Imai et al., 1997).

CHEMOKINE RECEPTOR FUNCTION IN HEALTH AND DISEASE The main function shared by chemokines and chemokine receptors is leukocyte chemotaxis, which, together with differential expression, allows for orchestration of specific leukocyte trafficking in vivo. Despite the redundancy in organization, there is increasing evidence from gene knockout and immunologic neutralization experiments for substantial specificity in chemokine and chemokine receptor function in vivo, affecting three main areas: organ development, susceptibility to infection, and inflammation (Table 3) (Gerard, 1999). This implies that chemokines which share leukocyte specificities and receptors which share chemokine specificities may not always be expressed at equivalent levels and in the same temporal and spatial context in vivo, and there is increasing experimental evidence for this (Amichay et al., 1996). Differential expression of combinations of chemokines or receptors could allow sequential action, directing leukocytes with high specificity to their in vivo targets (Foxman et al., 1997). In this scenario, agents that neutralize single chemokines or block single chemokine receptor subtypes could be very effective at terminating the entire signaling relay, and may be useful therapeutically in diseases where chemokine-dependent inflammation contributes to pathology. Chemokines and chemokine receptors can be loosely divided into three functional groups: immune, inflammatory, and an overlap group. Immune chemokine receptors, such as CXCR5 and CCR7, bind ligands that are constitutively expressed in a restricted manner and regulate basal leukocyte trafficking (Forster et al., 1996; Gunn et al., 1998a,b, 1999; Tang and Cyster, 1999; Saeki et al., 1999). This group of receptors regulates organization of the lymphoid system and determines the migration and position of T cells, B cells, and dendritic cells within specific areas of organized lymphoid tissue. In contrast, inflammatory receptors, such as CXCR1, CXCR2, CCR2, and CCR3, regulate emergency leukocyte trafficking to tissue sites of inflammation by binding ligands whose expression is less spatially restricted than immune chemokines, and instead is

Chemokine Receptors: Overview 1975 Table 3 Function of chemokine receptors in vivo: phenotypes associated with targeted gene disruptions in mice and naturally occurring inactivating mutations in humans Receptor

Viable?

Development

Major phenotypes

Mouse CXCR2

Yes

Abnormal

Neutrophil and B cell expansion in blood, lymph nodes, spleen, and bone marrow

Mouse CXCR4

No

Abnormal

Impaired neutrophil recruitment to i.p. thioglycollate Ventricular septal defect Impaired B cell lymphopoiesis Impaired bone marrow myelopoiesis Defective cerebellar and gastric vascular development Mouse CXCR5

Yes

Abnormal

Absent inguinal lymph nodes Absent or abnormal Peyer's patches Defective B cell trafficking and localization

Mouse CCR1

Yes

Normal

Impaired lung granuloma formation to Schistosoma mansoni eggs Reduced pancreatitis-induced pulmonary inflammation Increased susceptibility to Aspergillus fumigatus Abnormal TH1/TH2 cytokine balance in S. mansoni egg challenge Abnormal steady-state and induced trafficking and proliferation of myeloid progenitor cells

Mouse CCR2

Yes

Normal

Reduced monocyte recruitment after i.p. thioglycollate Reduced lung granuloma size to PPD challenge Abnormal TH1/TH2 cytokine balance in PPD challenge Increased susceptibility to Listeria Reduced atherogenesis

Mouse CCR5

Yes

Normal

Increased susceptibility to Listeria Increased susceptibility to LPS-induced endotoxemia Enhanced DTH reaction Increased humoral responses to T cell-dependent antigenic challenge

Human CCR5

Yes

NAD

Resistance to HIV-1 and AIDS

Human Duffy

Yes

NAD

Resistance to Plasmodium vivax form of malaria

highly temporally restricted by primary proinflammatory cytokines (e.g. IL-1 and TNF). Chemokines and chemokine receptors also regulate hematopoiesis. Analysis of knockout mice has shown that CXCR4 is required for B cell lymphopoiesis and bone marrow myelopoiesis, and that CXCR2, CCR1, and CCR2 regulate hematopoietic progenitor cell growth and distribution (Broxmeyer et al., 1996, 1999; Reid et al., 1999). In particular, CXCR2 is a negative regulator of myeloid progenitors, which may explain in part the massive expansion of neutrophils in mice lacking this receptor (Cacalano et al., 1994).

Chemokines and chemokine receptors may also play a role in T lymphocyte differentiation into TH1 and TH2 phenotypes, as suggested by studies of CCR1 and CCR2 knockout mice in schistosome egg challenge of the lung; however the mechanism underlying this is not yet clear (Boring et al., 1997; Gao et al., 1997). In addition to leukocytes, some chemokine receptors are also expressed on various other cell types, including erythrocytes, endothelial cells, neurons, and microglial cells of the brain (Hadley et al., 1994; Horuk et al., 1997; Gupta et al., 1998). The biological

1976 Philip M. Murphy significance of this is still undefined for most receptors, with the exception of CXCR4 (Tachibana et al., 1998; Zou et al., 1998). Consistent with expression in endothelial cells and neurons, genetic elimination of CXCR4 in mice causes defective neuronal cell migration in the cerebellum during development, a ventricular septal defect, and defective gastric vascular development. Another biologic process regulated by chemokines outside the hematopoietic system is angiogenesis. ELR-positive and -negative chemokines have been shown to have angiogenic and angiostatic activity, respectively, when injected in the rat cornea or overexpressed in animal models of cancer (Koch et al., 1992; Strieter et al., 1995). Whether this activity occurs physiologically is not yet known. Apart from roles in development, host defense, and inflammation, certain chemokine receptors may also function paradoxically and pathologically as promicrobial factors, the result of exploitation or subversion by specific microorganisms (McFadden et al., 1998; Pease and Murphy, 1998). The herpesvirus chemokine receptors mentioned earlier represent one mode of exploitation. The functions of these molecules are not yet understood, but possibilities include immune evasion through chemokine scavenging in the case of CMV US28 (Bodaghi et al., 1998; Vieira et al., 1998), and Kaposi's sarcoma tumorigenesis in the case of KSHV GPCR (Arvanitakis et al., 1997). Better understood are the subset of human chemokine receptors which are exploited by HIV as coreceptors, and function with CD4 as target cell entry factors. Of these, CCR5 and CXCR4 appear to be the most important in pathogenesis, and have distinct specificity for two major classes of HIV viruses, defined by leukocyte cytotropism. CCR5 is essential for efficient person-to-person HIV transmission and may also regulate the rate of disease progression, as revealed by analysis of the naturally occurring inactive allele CCR5
32 (reviewed in Moore et al., 1997; Doms and Peiper, 1997; Berger et al., 1999). Similarly, study of a defective Duffy allele affecting the promoter has revealed the obligate usage of normal Duffy by the protozoan Plasmodium vivax as a receptor for erythrocyte entry in the pathogenesis of malaria (Horuk et al., 1993; Horuk, 1994; Tournamille et al., 1995). Virally encoded chemokines have also been discovered, in several herpesviruses (e.g. HHV-8, mouse cytomegalovirus) and in molluscum contagiosum virus (MCV), a human poxvirus which causes the skin disease molluscum contagiosum. Interestingly, broad-spectrum chemokine receptor antagonist activity has been reported for several of these molecules, including vMIP-II of HHV-8 and MC148R of

MCV (Kledal et al., 1997; Damon et al., 1998), which suggests a mechanism for immune evasion by these viruses, and, reciprocally, argues for the importance of chemokines in antiviral host defense. Furthermore, various orthopoxviruses encode two structurally unique classes of secreted, broad-spectrum chemokine scavengers, one of which also binds IFN (reviewed in McFadden et al., 1998). Neither has structural homology to other proteins currently recorded in the public databases. To date, no naturally occurring mammalian chemokines have been identified that have chemokine receptor antagonist activity.

DEVELOPMENT OF CHEMOKINE RECEPTOR ANTAGONISTS Because of their specificity for leukocyte subsets, chemokine receptors are logical targets for drug development in human diseases characterized by inflammation. Over the past decade, substantial progress has been made in identifying targets and disease associations, and more recently in developing blocking strategies (Baggiolini and Moser, 1997). Animal models in which IL-8 has been neutralized immunologically have pointed to CXCR1 and CXCR2 as targets for diseases characterized by acute neutrophil-mediated inflammation, such as pustular psoriasis, glomerulonephritis, and ischemia±reperfusion injury, as may occur during angioplasty. Still, the available data do not discriminate specific roles of CXCR1 and CXCR2 in clinical disease. A small molecule antagonist specific for CXCR2 has been reported by White et al. (1998) from SmithKleinBeecham, but not yet evaluated preclinically. CCR3 is an attractive target in allergic inflammation and asthma because it appears to be the dominant chemokine receptor in human eosinophils, and is also expressed on basophils and a subset of TH2 lymphocytes. Consistent with this, genetic elimination of eotaxin, a major CCR3 ligand, in the mouse results in  50% reduction in airway inflammation after ovalbumin sensitization and challenge (Rothenberg et al., 1997). Extrapolation of this result to humans is restricted by the relative overexpression of CCR1 in mouse versus human eosinophils, the existence of other CCR3 ligands, and the potential for compensatory mechanisms in a nonconditional knockout. Direct CCR3 knockout will solve the second of these problems, but other strategies to determine the significance of CCR3 in disease are clearly needed. Using specific neutralizing antibodies, Kennedy et al. have found that acute and relapsing components

Chemokine Receptors: Overview 1977 of experimental allergic encephalomyelitis in mice are regulated by MIP-1 and MCP-1, respectively, suggesting potential roles of CCR1 and/or CCR5 and CCR2 in the corresponding phases of multiple sclerosis in humans (Kennedy et al., 1998). Selective small-molecule antagonists have been reported for all three of these receptors (Hesselgesser et al., 1998; Baba et al., 1999), but their effects in this and other disease models have not yet been published. Genetic knockouts in the mouse have revealed that CCR2 and its ligand MCP-1 contribute to the severity of atherosclerosis in dietary challenges (Boring et al., 1998), providing justification for investigation of specific antagonists in this disease. As alluded to above, direct proof of principle for the importance of specific chemokine receptors in clinical disease has been obtained only for CCR5 in AIDS and Duffy in vivax malaria. Indirect evidence has supported a role for CXCR4 in late stages of HIV infection (reviewed in Berger et al., 1999). Specific blocking agents, including small-molecule antagonists (Schols et al., 1997; Baba et al., 1999) have been identified for these receptors, but not yet tested in animal models or clinical trials. Blocking only one HIV coreceptor may be hazardous in HIV-infected persons, since the virus may simply develop resistance or the capacity to use another coreceptor more efficiently. A promising result for exploiting HIV coreceptors in vaccine development was recently reported (LaCasse et al., 1999), in which broad-spectrum neutralizing antibodies to HIV-1 were raised in mice immunized with CD4+ CCR5+ target cells crosslinked to HIV Env+ effector cells. Presumably, neutralizing epitopes are exposed transiently during coreceptor-promoted conformational changes in gp120 that occur transiently during the envelope± target cell membrane fusion reaction, and these are fixed by the crosslinking agent for stable and effective display to the immune system. Another potentially useful direction in therapeutics is to develop the viral chemokine antagonists and scavengers reviewed above, which may be best suited as topical agents or single administration agents for acute inflammation, such as occurs after angioplasty.

CONCLUSION Although additional chemokines and chemokine receptors will probably continue to be identified for some time, adding to what is already a system of enormous complexity, there is already enough known to discern important patterns of specificity and activity, as described in this overview. An area which

remains relatively poorly developed and in which advances may have the greatest impact on further knowledge of the biology of the system is in chemokine receptor pharmacology. At present the system can be viewed as extremely rich in endogenous agonists, but equally poor in selective antagonists. Filling this void may facilitate biological experiments that cannot be done easily, or at all, using genetic and immunologic approaches currently in use, and lead to new treatments of human disease.

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