AU2002213318B2 - CD38 modulated chemotaxis - Google Patents

Description

BAKER & BOTTS, L.L.P.

30 ROCKEFELLER PLAZA

NEW YORK, NEW YORK 10112

TO ALL WHOM IT MAY CONCERN:

Be it known that WE, Frances E. Lund and Troy D. Randall, citizens of the United States and Santiago Partida-Sanchez, a citizen of Mexico, whose post office addresses are PO Box 59, Saranac Lake, N.Y. have invented an improvement in

CD38 MODULATED CHEMOTAXIS

of which the following is a

SPECIFICATION

1. INTRODUCTION

[0001] The present invention relates to methods for modulating the migratory activity of cells

expressing CD38 for the treatment of disorders including, but not limited to, inflammation, ischemia,

asthma, autoimmune disease, diabetes, arthritis, allergies, infection with pathogenic organisms, such

as parasites, and transplant rejection. Such cells include, for example, neutrophils, lymphocytes,

eosinophils, macrophages and dentritic cells. The invention further relates to drug screening assays

designed to identify compounds that modulate the ADP-ribosyl cyclase activity of CD38 and the use

of such compounds in the treatment of disorders involving CD38 modulated cell migration.

Additionally, the invention relates to the isolation and characterization of a CD38 homologue from the parasitic flatworm, Schistosoma mansoni. The identification of such a homologue, referred to herein as SM38, provides compositions and assays designed to screen for related enzymes in pathogenic organisms as well as compositions and assays to screen for compounds that modulate the activity and/or expression of SM38. Such compounds can be used to treat pathogenic disorders resulting from infection with such parasites. The invention is based on the discoveries that CD38 ADP-ribosyl cyclase activity is required for chemotaxis and that S. mansoni expresses a CD38 homologue that can regulate calcium responses in the parasite.

2. BACKGROUND OF INVENTION [0002] Hematopoietically-derived cells, including cells such as neutrophils, monocytes, dendritic cells, eosinophils and lymphocytes, are important cellular mediators of the inflammatory response and respond to soluble inflammatory mediators by migration to the site of tissue injury or infection where the newly arrived cells perform their effector functions.

[0003] Neutrophils which represent 40-50 % of the circulating leukocyte population are particularly important to both immunity and inflammation. Neutrophils are normally quiescent cells but upon stimulation can mediate a variety of different inflammatory activities. A large number of different agents are capable of activating neutrophils and this activation is normally mediated by binding of the activating agent to specific receptors expressed on the surface of neutrophils. Once activated, the neutrophils are capable of binding to endothelial cells and migrating to the site of tissue damage, a pathogen or a foreign material. Similarly, eosinophils are also potent inflammatory effector cells, although these cells are most often associated with allergic diseases such as asthma.

Like neutrophils, eosinophils have a potent armory of proinflammatory molecules that can initiate and maintain inflammatory responses.

[0004] Once at the inflammatory site, recruited cells such as eosinophils and neutrophils induce

further inflammation by releasing inflammatory products and recruiting other hematopoietically-

derived cells to the site, hi some cases, the inflammatory response mediated by the specifically

recruited hematopoietically-derived cells protects the host from morbidity or mortality by eliminating

the infectious agent. In other cases {i.e., autoimmunity, ischemia/reperfusion, transplantation,

allergy), the inflammatory response further damages the tissue resulting in pathology. Thus, agents

which alter inflammation or recruitment of cells may be useful in controlling pathology.

[0005] Although CD38 expression was at first believed to be restricted to cells of the B cell

lineage, subsequent experiments by a number of groups have demonstrated that CD38 is widely

expressed on both hematopoietic and non-hematopoietically-derived cells. Homologues of CD38

have also been found to be expressed in mammalian stromal cells (Bst-1) and in cells isolated from

the invertebrate Aplysia californica (ADP-ribosyl cyclase enzyme) (Prasad GS, 1996, nature

Structural Biol 3:957-964)

[0006] More recently, CD38 was shown to be a multifunctional ecto-enzyme with NAD⁺

glycohydrolase activity and ADP-ribosyl cyclase activity, enabling it to produce nicotinamide, ADP- ribose (ADPR), cyclic- ADPR (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP)

from its substrates NAD⁺ andNADP (Howard et al., 1993 Science 252:1056-1059; Lee et al., 1999

Biol. Chem. 380;785-793). Two of the metabolites produced by CD38, cADPR and NAADP, have been shown to induce the release of intracellular calcium in cells isolated from tissues of plants,

invertebrates and mammals, suggesting that these metabolites may be global regulators of calcium

responses (Lee et al., 1999 Biol. Chem. 380;785-793).

[0007] Both cADPR and NAADP are known to induce calcium release from calcium stores that

are distinct from those controlled by IP receptors (Clapper, DL et al., 1987, J. Biological Chem.

262:9561-9568). Instead, cADPR is believed to regulate calcium release from ryanodine receptor

regulated stores, as agonists of ryanodine receptors sensitize cADPR mediated calcium release and

antagonists of ryanodine receptors block cADPR dependent calcium release (Galione A et al., 1991,

Science 253:143-146). Thus, it has been proposed that cADPR is likely to regulate calcium

responses in tissues such as muscle and pancreas where ryanodine receptors are expressed.

Interestingly, it was recently shown that the muscle fibers of the parasitic flatworm, S. mansoni,

express ryanodine receptors and that agonists of ryanodine receptors such as caffeine can induce

intracellular calcium release and muscle contraction in the parasite (Day et al, 2000 Parasitol

120:417-422; Silva et al., 1998, Biochem. Pharmacol 56:997-1003). hi mammalian smooth muscle

cells, the calcium release in response to acetylcholine can be blocked not only with ryanodine

receptor antagonists, but also with specific antagonists of cADPR such as 8-NH²-cADPR or 8-Br-

cADPR (Guse, AH, 1999, Cell. Signal. 11:309-316).

[0008] These findings, as well as others, indicate that ryanodine receptor agonists/antagonists

icnluding cADPR can regulate calcium responses in cells isolated from species as diverse as

helminths to mammals, however, it is unclear whether ADP-ribosyl cyclase enzymes such as CD38 or SM38 are required for the production of c ADPR in vivo. Additionally, there has been no direct evidence to link CD38 enzyme activity with downstream responses such as calcium release, proliferation, apoptosis, migration or other effector functions. Thus, despite the high level expression of CD38 on many cell types, no clear defining role for CD38 enzyme activity in immune

responses has been established.

3. SUMMARY OF THE INVENTION

[0009] The present invention relates to methods for modulating the migratory activity of cells expressing CD38 involving the administration of agonists or antagonists of CD38 enzyme activity, and the cADPR mediated signal transduction pathway, including small molecules, large molecules, and antibodies. The invention also provides for compounds and nucleotide sequences that can be

used to modulate CD38 gene expression.

[0010] The present invention further relates to the isolation and characterization of a CD38 homologue from the parasitic flatworm Shistosoma mansoni, herein referred to as SM38. The identification of such a homologue provides compositions and assays designed to screen for related enzymes in pathogenic micro-organisms (such as helminths) as well as compositions and assays to screen for compounds that modulate the activity of SM38. Such compounds can be used to treat pathogenic disorders resulting from infection with such pathogenic micro-organisms. [0011] The invention relates to assays designed to screen for compounds that modulate the

enzymatic activity of CD38 and/or SM38 (CD38/SM38), i.e., compounds that act as agonists and

antagonists of CD38 enzyme activity. In addition, the screens of the invention may be used to

identify substrates of CD38/SM38 that are converted into antagonists or agonists of signal transduction pathways involving cADPR. The screens of the invention also maybe used to directly

identify agonists and antagonists of signal transduction pathways involving cADPR.

[0012] The invention also relates to assays designed to screen for compounds that modulate

CD38/SM38 gene expression. For example, cell-based assays can be used to screen for compounds

that modulate CD38/SM38 transcription such as compounds that modulate expression, production

or activity of transcription factors involved in CD38/SM38 gene expression; antisense and ribozyme

polynucleotides that modulate translation of CD38/SM38 mRNAs and polynucleotides that form

triple helical structures with the CD38/SM38 regulatory regions and inhibit transcription of the

CD38/SM38 gene.

[0013] Identified compounds may be used in the treatment of disorders where the migratory activity of CD38-expresssing cells, such as hematopoietically-derived cells, contributes to the

development of such disorders. Such disorders include, but are not limited to inflammation,

ischemia, asthma, autoimmune disease, diabetes, arthritis, allergies or transplant rejection where

inhibition of migratory activity using, for example, CD38 antagonists would be desired. In contrast,

in subjects infected with pathogenic microorganisms or immunosuppressed subjects it may be

desirable to induce the migratory activity of hematopoietically-derived cells using, for example, agonists of CD38. Additionally, identified compounds may be used to treat pathogenic disorders

resulting from infection with pathogenic micro-organisms expressing SM38 or structurally related

homologous proteins.

4. BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1. Normal Cellular Response to Chemoattractant Signaling. (1) Chemoattractant

binds to receptor and initiates signaling. (2) CD38 hydrolyzes NAD and produces cADPR, which

facilitates Ca2+ release from internal stores. (3) Ca2+ is released from cADPR-controlled internal

stores which activates external Ca2+ channel. (4) Extracellular Ca2+ flows into the cell and allows

migration.

[0015] Figure 2. Inhibitors of cADPR Production by CD38 Prevent Capacitative Ca2+ Entry and

Chemoattractant Induced Migration (Screens will identify such compounds). (1) Chemoattractant

binds to receptor and initiates signaling. (2) Inhibitor of CD38 prevents either hydrolysis of NAD

(enzyme is inactive and no products are made) or specifically inhibits production of cADPR (blocks

ADP-ribosyl cyclase activity, but enzyme may not be inactive). (3) Lack of cADPR results in no

cADPR-mediated Ca2+ release from internal stores. (4) No capacitative Ca2+ influx and no

migration.

[0016] Figure 3. Proteins that Regulate CD38 Enzyme Activity (Screens will identify compounds

that activate or inactivate these proteins). (1) Chemoattractant binds to receptor and initiates signaling. (2) Protein X modifies CD38 and inactivates CD38 enzyme activities. (3) Lack of cADPR results in no cADPR-mediated Ca2+ release from internal stores. (4) No capacitative Ca2+ influx

and no migration.

[0017] Figure 4. Proteins that Regulate CD38 Expression (Screens will identify compounds that activate or inactivate these proteins). (1) Chemoattractant binds to receptor and initiates signaling. (2) Protein X represses CD38 gene transcription. (3) Lack of CD38 results in absence of cADPR which results in no cADPR-mediated Ca2+ release from internal stores. (4) No capacitative Ca2+ influx and no migration.

[0018] Figure 5. Alternate Substrates for CD38 may generate inhibitors of cADPR and prevent capacitative Ca2+ release (Screens will identify such compounds). (1) Chemoattractant binds to receptor and initiates signaling. (2) CD38 hydrolyzes modified substrate (8-BrNAD, for example)and produces modified product (8-Br-cADPR, for example) (3) Modified product competitively or non-

competitively inhibits cADPR induced Ca2+ release from internal stores. (4) No capacitative Ca2+ influx and no migration.

[0019] Figure 6. Inhibitors of cADPR binding block capacitative Ca2+ influx. (1) Chemoattractant binds to receptor and initiates signaling (Screens will identify such compounds). (2) CD38 hydrolyzes NAD and produces cADPR. (3) Inhibitor of cADPR (8-Br cADPR) competitively or non-competitively blocks cADPR induced Ca2+ release from internal stores. (4) No capacitative Ca2+ influx and no migration. [0020] Figure. 7. CD38KO mice are more susceptible to S. pneumomae infection, (a) C57BL/6

WT (open circles) and CD38KO (filled circles) mice were infected intra-tracheally with two doses

of S. pneumoniae. The survival of infected animals was monitored over the next 4 days, (b) WT

mice that had been irradiated and reconstituted with WT bone marrow (open squares) or CD38KO

bone marrow (filled squares) were infected with two doses of S. pneumoniae and monitored for four

days. The data are representative of at least 5 independent experiments. n=10 mice/group, (c) WT

or Rag-2 KO (open bars) and CD38KO or CD38-Rag-2 double KO (filled bars) mice were infected

intra-tracheally with S. pneumoniae and bacterial titers in lung and peripheral blood were determined

at 12 hours post-infection. The data are representative of 3 independent experiments. n= 0

mice/group. *P<0.001; Student's t test.

[0021] Figure. 8. CD38KO neutrophils are not recruited to the infection site and are unable to

chemotax toward bacterially-derived chemoattractants. WT and CD38KO mice were infected intra-

tracheally with S. pneumoniae, and the cellular infiltrate in the airways was collected and counted

(panel a, WT=open bars and CD38KO=closed bars) at multiple timepoints post-infection, (b) The

identity and frequency of the infiltrating cells in the lungs of infected WT and CD38KO mice was

determined by microscopic examination (400X magnification) and counting of Diff-Quick stained

cytocentrifuge preparations, (c) Differential cell counts in the lung lavage of WT (open bars) and

CD38KO (closed bars) mice are presented as the mean number of cells x 10⁶ ± (SE). Similar results

were obtained in 5 independent experiments. n=5 mice/group/timepoint. *P<0.01 **P=0.01;

Student's t Test, (d) Purified bone marrow neutrophils from WT (open bars) and CD38KO mice (filled bars) were tested for their ability to migrate in response to medium, fMLP or IL-8 in a conventional transwell checkerboard chemokinesis/chemotaxis assay. The number of cells migrating to the bottom chamber of the transwell in the absence of any stimulation was not significantly different between CD38KO and WT neutrophils and ranged from 1500-2300 cells (not shown). The number of neutrophils migrating in response to equivalent concentrations of stimuli in both chambers (chemokinesis) and the number of neutrophils migrating in response to a chemotactic gradient (chemotaxis) is shown. The values shown are the mean ± S.E. of four different

experiments. *P <0.001; Student's t Test.

[0022] Figure 9. CD38 expressing neutrophils produce cADPR and release intracellular calcium in response to cADPR and ryanodine. (a) Bone marrow, peripheral blood and peritoneal cavity cells

were isolated from WT and CD38KO mice or WT and CD38KO mice that received an intraperitoneal injection of thioglycollate 12 hrs previously. CD38 expression on the Mac-1 GR-1 neutrophils was analyzed by flow cytometry. Expression of CD38 on WT neutrophils (solid line histogram) and CD38KO neutrophils (dotted line histogram) is shown, (b) CD 15+ human peripheral blood neutrophils were assessed for CD38 expression by staining with anti-CD38 mAb (filled histogram) or an isotype control Ab (dotted line), (c) Cyclase activity was measured in WT and CD38KO bone marrow neutrophils. WT or CD38KO neutrophils were incubated alone (WT=circles and CD38KO=squares) or in the presence of NGD (WT=triangles and CD38KO=diamonds) for 10 minutes. The accumulation of the product, cGDPR, was measured fluorometrically. (d) RyR3 mRNA expression levels were determined by RT-PCR. cDNA was isolated from purified WT bone marrow neutrophils (PMN) or brain tissue. The amount of input cDNA is indicated, (e-g)

Intracellular free calcium levels were measured by FACS in Fluo-3/Fura Red loaded bone marrow

neutrophils. (e) Neutrophils were permeabilized with digitonin and then stimulated with ryanodine

in the presence (orange line) or absence (blue line) of ruthenium red. (f) Neutrophils were

permeabilized in digitonin and then stimulated with cADPR (blue line), heat inactivated cADPR

(green line) or 8-Br-cADPR + cADPR (red line), (g) Neutrophils were stimulated with thapsigargin

(blue line) or thapsigargin + 8-Br-cADPR (red line). All data in panels e-g are representative of at

least three independent experiments.

[0023] Figure 10. CD38 catalyzed cADPR regulates intracellular calcium release, extracellular

calcium influx and chemotaxis in neutrophils. (a-c) Intracellular free calcium levels were measured

by FACS in Fluo-3/Fura Red loaded bone marrow neutrophils. (a) CD38KO (red line) and WT

(blue line) neutrophils were stimulated with fMLP or IL-8 in calcium-free buffer, (b) CD38KO (red

line) and WT (blue line) neutrophils were stimulated with fMLP or IL-8 in calcium-containing

buffer, (c) CD38KO (red line) and WT (blue line) neutrophils were preincubated in calcium-

containing medium ± 8-Br-cADPR and then stimulated with fMLP or IL-8. All data in panels a-c

are representative of at least five independent experiments, (d) WT neutrophils were pre-incubated

with medium, EGTA or 8-Br-cADPR and then placed in the top chamber of a transwell that

contained fMLP or IL-8 in the bottom chamber. The cells that migrated to the bottom chamber in

response to the chemotactic gradient were collected and enumerated by flow cytometry. Values

shown are mean ± S.E. from three separate experiments with three wells/experimental condition. **P=0.008; Maim Whitney Rank Sum Test.

[0024] Figure 11. An NAD+ analogue regulates calcium influx and chemotaxis in fMLP-activated

neutrophils. (a) Dye-loaded purified bone marrow neutrophils from WT mice were preincubated in

medium (blue line) or increasing concentrations of N(8-Br-A)D+ (red line) and then stimulated with

fMLP. Changes in intracellular calcium levels were measured by flow cytometry. The data are

representative of three independent experiments, (b-c) WT (left panel) and CD38KO (right panel)

neutrophils were preincubated with medium (filled bars) or N(8-Br-A)D+ (open bars) and then

placed in the top chamber of a transwell which contained fMLP (panel b) or IL-8 (panel c) in the

bottom chamber. The cells that migrated to the bottom chamber in response to the chemotactic

gradient were collected and enumerated by flow cytometry. Values shown are mean ± S.E. from

three separate experiments with three wells/experimental condition. *P<0.001 Student's t Test.

[0025] Figure 12 A-B. The recruitment of neutrophils and eosinophils to the lungs in a model of

allergic asthma is impaired in CD38 KO mice. Naive CD4 T cells from WT C57BL/6 mice or OVA-

primed CD4 T cells from WT C57BL/6 mice were transferred to either WT C57BL/6 mice or to

CD38KO-C57BL/6 mice as indicated. Recipient mice were subsequently challenged on 7

consecutive days by intratracheal instillation of 10 μg OVA in PBS. Neutrophils (A) and eosinophils

(B) in the lung lavage on the eighth day after initial challenge were enumerated by microscopic

examination (400X) of Diff-Quick stained cytocentrifuge preparations

[0026] Figure 13. Comparison of S. mansoni SM38 cDNA with S. mansoni ESTs. The SM38 cDNA was isolated and cloned from an S. mansoni cDNA library as described in methods. The

cDNA for SM38 includes 5' untranslated sequence (gray box), an initiation methionine (underlined),

an open reading frame encoding a 303 amino acid protein (clear box), a stop codon (underlined), 3'

untranslated sequence (gray box) and a poly-adenylation site (underlined). The full-length cDNA

was compared to published S. mansoni EST sequences and three separate EST sequences were found

that were identical to portions of the SM38 sequence. The SM38 cDNA includes 421 base pairs of

unique sequence (70 bp 5' untranslated and 351 bp of open reading frame) not found in any public

database.

[0027] Figure 14. Translation of SM38 cDNA. The SM38 cDNA was translated in all reading

frames and an open reading frame of 303 amino acids was identified. The initiation codon is located

at nucleotide position 71-73 and the termination codon is found at nucleotide position 980-983.

[0028] Figure 15. SM38 is homologous to Aplysia ADP-ribosyl cyclase and human CD38 cyclase.

The protein sequence of SM38 was aligned with the protein sequences for Aplysia ADP-ribosyl

cyclase (part a) and human ADP-ribosyl cyclase CD38 (part b). A high degree of homology (boxed

residues) was observed with 21% identity between the Aplysia protein and SM38 and 23% identity

between human CD38 and SM38. The conserved 10 cysteine residues present in all members of the

cyclase protein family are also present in SM38 (shaded boxes). The two additional cysteines found in CD38 (underlined), but not in Aplysia, are also lacking in SM38. However, the SM38 protein

contains two additional cysteine residues that are unique and are not found in either CD38 or Aplysia

cyclase (underline). Most importantly, the active site catalytic residues identified for CD38 and Aplysia enzyme (starred residues) are also present in SM38.

[0029] Figure 16. SM38 is a soluble protein. The protein sequence of SM38 was examined to

determine if the protein is a type-II membrane bound protein like CD38, a soluble protein like the

Aplysia cyclase, a GPI-linked protein like other cyclase family proteins, or a secreted protein. The

conserved enzyme domain (see previous figure) is shaded. SM38 contains only 22 amino acids 5'

of the enzyme domain. These 22 amino acids are not hydrophobic, thus, no leader sequence 5' of

the enzyme domain could be identified, indicating that SM38 is not secreted or GPI-linked.

Additionally, no 5' transmembrane domain could be identified, indicating that SM38 is not a type-II

membrane protein. Therefore, SM38 is most likely a soluble cytoplasmic protein like Aplysia

cyclase.

[0030] Figure 17. Reverse translation of SM38. The 303 amino acid coding region of SM38 was

reverse-translated to identify a degenerate DNA sequence that would encode the SM38 protein.

5. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to methods for modulating the migratory activity of cells

involving the regulation of CD38 ADP-ribosyl cyclase activity. The invention is based on the

discovery that granulocytes such as neutrophils and eosinophils from CD38KO mice cannot be

efficiently recruited to sites of inflammation and infection in the body. The invention is based on

the discovery that although CD38 ADP-ribosyl cyclase activity is not essential for the initial activation of granulocytes such as neutrophils, it is critically important in regulating neutrophil

chemotaxis both in vivo and in vitro, hi particular, cADPR, a product of CD38 ADP-ribosyl cyclase

activity, is required to induce calcium release from calcium stores present within neutrophils. The

release of calcium from this specialized store is necessary for activation and opening of plasma

membrane channels resulting in a capacitative influx of calcium that subsequently mediates the

direct migration of neutrophils toward chemoattractants and/or inflammatory products.

[0032] The present invention encompasses screening assays designed for the identification of

modulators, such as agonists and antagonists, of CD38 enzyme activity and/or modulators of cADPR

dependent calcium responses and chemotaxis. The invention further relates to the use of such

modulators in the treatment of disorders based on the CD38 controlled migratory activity of cells to

chemoattractants and inflammatory products. Such disorders include, but are not limited to,

inflammation, ischemia, autoimmune disease, asthma, diabetes, arthritis, allergies, infections and

organ transplant rejection.

[0033] The present invention also relates to the identification, isolation and characterization of the

CD38 homologue, SM38, from the parasite S. mansoni. The invention encompasses screening

assays to identify related enzymes in other pathogenic micro-organisms, such as helminths, as well

as compositions and assays to screen for compounds that modulate the activity and expression of

SM38. The invention further relates to the use of such modulators to treat pathogenic disorders in

animals and humans infected with organisms expressing SM38 or structurally related molecules. [0034] Various aspects of the invention are described in greater detail in the subsections below.

5.1. THE SM38 GENE

[0035] The cDNA sequence and deduced amino acid sequence of S. mansoni SM38 is shown in

Figure 14 (ATCC Deposit No: ). The SM38 cDNA was translated in all reading frames and an open

reading frame of 303 amino acids was identified. The initiation codon is located at nucleotide

position 71 and the termination codon is found at nucleotide position 981.

[0036] The SM38 nucleotide sequences of the invention include: (a) the DNA sequences shown

in Figure 14; (b) a nucleotide sequences that encodes the amino acid sequence shown in Figure 14;

(c) any nucleotide sequence that (i) hybridizes to the nucleotide sequence set forth in (a) or (b) under

stringent conditions, ejg., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl

sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et

al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, ie,

and John Wiley & sons, h e, New York, at p. 2.10.3) and (ii) encodes a functionally equivalent gene

product; and (d) any nucleotide sequence that hybridizes to a DNA sequence that encodes the amino

acid sequence shown in Figure 14 under less stringent conditions, such as moderately stringent

conditions, e^, washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel et al., 1989 supra), yet which still

encodes a functionally equivalent SM38 gene product. Functional equivalents of the SM38 protein

include naturally occurring SM38 present in species other than S. mansoni. The invention also

includes degenerate variants of sequences (a) through (d). The invention also includes nucleic acid

molecules, that may encode or act as SM38 antisense molecules, useful, for example, in SM38 gene regulation (for and/or as antisense primers in amplification reactions of SM38 gene nucleic acid

sequences).

[0037] In addition to the SM38 nucleotide sequences described above, homologs of the SM38 gene

present in other species can be identified and readily isolated, without undue experimentation, by

molecular biological techniques well known in the art. For example, cDNA libraries, or genomic

DNA libraries derived from the organism of interest can be screened by hybridization using the

nucleotides described herein as hybridization or amplification probes.

[0038] The invention also encompasses nucleotide sequences that encode mutant SM38s, peptide

fragments of the SM38, truncated SM38, and SM38 fusion proteins. These include, but are not

limited to nucleotide sequences encoding polypeptides or peptides corresponding to the cyclase

domain of SM38 or portions of this domain; truncated SM38s in which the domain is deleted, e.g.,

a functional SM38 lacking all or a portion of the cyclase region. Certain of these truncated or mutant

SM38 proteins may act as dominant-negative inhibitors of the native SM38 protein. Nucleotides

encoding fusion proteins may include but are not limited to full length SM38, truncated SM38 or peptide fragments of SM38 fused to an unrelated protein or peptide such as an enzyme, fluorescent

protein, luminescent protein, etc., which can be used as a marker.

[0039] SM38 nucleotide sequences may be isolated using a variety of different methods known

to those skilled in the art. For example, a cDNA library constructed using RNA from cells or tissue

known to express SM38 can be screened using a labeled SM38 probe. Alternatively, a genomic library may be screened to derive nucleic acid molecules encoding the SM38 protein. Further, SM38

nucleic acid sequences may be derived by perfonning PCR using two oligonucleotide primers

designed on the basis of the SM38 nucleotide sequences disclosed herein. The template for the

reaction may be cDNA obtained by reverse transcription of mRNA prepared from cell lines or tissue

known to express SM38.

[0040] The invention also encompasses (a) DNA vectors that contain any of the foregoing SM38

sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any

of the foregoing SM38 sequences operatively associated with a regulatory element that directs the

expression of the SM38 coding sequences; (c) genetically engineered host cells that contain any of

the foregoing SM38 sequences operatively associated with a regulatory element that directs the

expression of the SM38 coding sequences in the host cell; and (d) transgenic mice or other

organisms that contain any of the foregoing SM38 sequences. As used herein, regulatory elements

mclude but are not limited to inducible and non-inducible promoters, enhancers, operators and other

elements known to those skilled in the art that drive and regulate expression.

5.1.2. SM38 PROTEINS AND POLYPEPTIDES

[0041] SM38 protein, polypeptides and peptide fragments, mutated, truncated or deleted forms of

the SM38 and/or SM38 fusion proteins can be prepared for a variety of uses, including but not

limited to the generation of antibodies, the identification of other cellular gene products involved in

the regulation of SM38 activity, and the screening for compounds that can be used to modulate the activity of SM38. [0042] Figure 14 shows the deduced amino acid sequence of the SM38 protein. The SM38 amino acid sequences of the invention include the amino acid sequence shown in Figure 14. Further, SM38s of other species are encompassed by the invention. In fact, any SM38 protein encoded by the SM38 nucleotide sequences described above is within the scope of the invention.

[0043] The invention also encompasses proteins that are functionally equivalent to the SM38 encoded by the nucleotide sequences described in Section 5.1, as judged by any of a number of criteria, including but not limited to the ability to catalyze the production of the calcium mobilizing second messenger, cADPR and thereby regulate calcium response. Such functionally equivalent SM38 proteins include but are not limited to proteins having additions or substitutions of amino acid residues within the amino acid sequence encoded by the SM38 nucleotide sequences described, above, in Section 5.1, but which result in a silent change, thus producing a functionally equivalent gene product.

[0044] Peptides corresponding to one or more domains of SM38 as well as fusion proteins in which the full length SM38, a SM38 peptide or a truncated SM38 is fused to an unrelated protein are also within the scope of the invention and can be designed on the basis of the SM38 nucleotide and SM38 amino acid sequences disclosed herein. Such fusion proteins include fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker function.

[0045] While the SM38 polypeptides and peptides can be chemically synthesized (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY.), large polypeptides derived from SM38 and the frill length SM38 itself may be advantageously produced

by recombinant DNA technology using techniques well known in the art for expressing a nucleic

acid containing SM38 gene sequences and/or coding sequences. Such methods can be used to

construct expression vectors containing the SM38 nucleotide sequences described in Section 5.1 and

appropriate transcriptional and translational control signals. These methods include, for example,

in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See,

for example, the techniques described in Sambrook et al., 1989, supra, and Ausubel et al., 1989,

supra).

[0046] A variety of host-expression vector systems may be utilized to express the SM38 nucleotide

sequences of the invention. Where the SM38 peptide or polypeptide is expressed as a soluble

derivative and is not secreted, the peptide or polypeptide can be recovered from the host cell.

Alternatively, where the SM38 peptide or polypeptide is secreted the peptide or polypeptides may

be recovered from the culture media. Purification or enrichment of the SM38 from such expression

systems can be accomplished using appropriate detergents and lipid micelles and methods well

known to those skilled in the art. Such engineered host cells themselves may be used in situations

where it is important not only to retain the structural and functional characteristics of the SM38, but

to assess biological activity, i.e., in drug screening assays.

[0047] The expression systems that may be used for purposes of the invention include but are not

limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid

or cosmid DNA expression vectors containing SM38 nucleotide sequences; yeast transformed with recombinant yeast expression vectors containing SM38 nucleotide sequences or mammalian cell

systems harboring recombinant expression constructs containing promoters derived from the genome

of mammalian cells or from mammalian viruses.

[0048] Appropriate expression systems can be chosen to ensure that the correct modification,

processing and sub-cellular localization of the SM38 protein occurs. To this end, host cells which

possess the ability to properly modify and process the SM38 protein are preferred. For long-term,

high yield production of recombinant SM38 protein, such as that desired for development of cell

lines for screening purposes, stable expression is preferred. Rather than using expression vectors

which contain origins of replication, host cells can be transformed with DNA controlled by

appropriate expression control elements and a selectable marker gene, i.e., tk, hgprt, dhfr, neo, and

hygro gene, to name a few. Following the introduction of the foreign DNA, engineered cells may

be allowed to grow for 1-2 days in enriched media, and then switched to a selective media. Such

engineered cell lines may be particularly useful in screening and evaluation of compounds that

modulate the endogenous activity of the SM38 gene product.

5.1.3. TRANSGENIC ANIMALS

[0049] The SM38 gene products can also be expressed in transgenic animals. Animals of any

species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and

non-human primates, e^., baboons, monkeys, and chimpanzees may be used to generate SM38 transgenic animals. [0050] Any technique known in the art may be used to introduce the SM38 transgene into animals

to produce the founder lines of transgenic animals. Such techniques include, but are not limited to

pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus

mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci. USA

82:6148-6152); gene targeting in embryonic stem cells (Thompson et al, 1989, Cell, 56:313-321);

electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene

transfer (Lavifrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon,

1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which is incorporated by reference herein

in its entirety.

[0051] The present invention provides for transgenic animals that carry the SM38 transgene in all

their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic

animals. The transgene may also be selectively introduced into and activated in a particular cell type

by following, for example, the teaching of Lasko et al., (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci.

USA 89:6232-6236). The regulatory sequences required for such a cell-type specific activation will

depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

When it is desired that the SM38 transgene be integrated into the chromosomal site of the

endogenous SM38 gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous SM38 gene are

designed for the purpose of integrating, via homologous recombination with chromosomal

sequences, into and disrupting the function of the nucleotide sequence of the endogenous SM38 gene. [0052] Once transgenic animals have been generated, the expression of the recombinant SM38

gene may be assayed utilizing standard techniques. Initial screening may be accomplished by

Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of

the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the

transgenic animals may also be assessed using techniques which include but are not limited to

Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and

RT-PCR. Samples of SM38 gene-expressing tissue may also be evaluated immunocytochemically

using antibodies specific for the SM38 transgene product.

5.4. ANTIBODIES TO SM38 PROTEINS

[0053] Antibodies that specifically recognize one or more epitopes of SM38, or epitopes of

conserved variants of SM38, or peptide fragments of SM38 are also encompassed by the invention.

Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs),

humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments,

fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-

binding fragments of any of the above.

[0054] The antibodies of the invention may be used, for example, in conjunction with compound

screening schemes, as described, below, for the evaluation of the effect of test compounds on expression and/or activity of the SM38 gene product.

[0055] For production of antibodies, various host animals may be immunized by injection with a SM38 protein, or SM38 peptide. Such host animals may include but are not limited to rabbits,

mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological

response, depending on the host species, including but not limited to Freund' s (complete and

incomplete), mineral gels such as aluminum hydroxide, surface active substances such as

lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,

dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and

Corynebacterium parvum .

[0056] Polyclonal antibodies comprising heterogeneous populations of antibody molecules, may

be derived from the sera of the immunized animals. Monoclonal antibodies maybe obtained by any

technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein,

(1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique

(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc, Natl. Acad. Sci. USA

80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And

Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin

class including IgG, IgM, IgE, IgA, IgD and any subclasses thereof. The hybridoma producing the

mAb of this invention may be cultivated in vitro or in vivo. Production of high titres of Mabs in vivo

makes this the presently preferred method of production.

[0057] hi addition, techniques developed for the production of "chimeric antibodies" by splicing

the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used (Morrison et al.,

1984, Proc. Natl Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda

et al. 1985, Nature 314: 452-454). Alternatively, techniques developed for the production of

humanized antibodies (U.S. Patent No. 5,585,089) or single chain antibodies (U.S. Patent No.

4,946,778 Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl Acad. Sci USA, 85:

5879-5883; and Ward et al., 1989, Nature 334: 544-546) may be used to produce antibodies that

specifically recognize one or more epitopes of SM38.

5.2. SCREENING ASSAYS FOR COMPOUNDS USEFUL IN_. MODULATING THE ACTIVITY OF CD38/SM38

[0058] The present invention relates to screening assay systems designed to identify compounds

or compositions that modulate CD38/SM38 enzyme activity, cADPR mediated signal transduction,

or CD38/SM38 gene expression, and thus, may be useful for modulation of cell migration or

treatment of infection.

5.2.1. RECOMBINANT EXPRESSION OF CD38

[0059] For purposes of developing screening assays designed to identify compounds or

compositions that modulate CD38/SM38 activity it may be necessary to recombinantly express the

CD38/SM38 proteins. The cDNA sequence and deduced amino acid sequence of CD38 has been

characterized from several species including human, murine and rat as described in Jackson, D.G.

et al., 1990, J. Immunol. 151:3111-3118; Koguma, T. et al., 1994, Biochim Biophys Acta 1224:160-

162 and Harada N et al., 1993, J Immunol 151:3111-3118, incorporated herein by reference. In addition, the cDNA and deduced amino acid sequence of Shistosoma mansoni, as described herein

maybe utilized to recombinantly express the CD 38 homologue, SM38, protein.

[0060] CD38/SM38 nucleotide sequences may be isolated using a variety of different methods

known to those skilled in the art. For example, a cDNA library constructed using RNA from a tissue

known to express CD38/SM38 can be screened using a labeled CD38/SM38 probe. Alternatively,

a genomic library may be screened to derive nucleic acid molecules encoding the CD38/SM38

protein. Further, CD38/SM38 nucleic acid sequences may be derived by performing a polymerase

chain reaction (PCR) using two oligonucleotide primers designed on the basis of known CD38/SM38

nucleotide sequences. The template for the reaction may be cDNA obtained by reverse transcription

of mRNA prepared from cell lines or tissue known to express CD38/SM38 .

[0061] CD38/SM38 protein, polypeptides and peptide fragments, mutated, truncated or deleted

forms of CD38/SM38 and/or CD38/SM38 fusion proteins can be prepared for a variety of uses, including but not limited to the generation of antibodies, the identification of other cellular gene

products involved in the regulation of CD38/SM38 mediated cell migration, and the screening for

compounds that can be used to modulate cell migration. CD38/SM38 fusion proteins include fusions

to an enzyme, fluorescent protein, a polypeptide tag or luminescent protein which provide a marker

function.

[0062] While the CD38/SM38 polypeptides and peptides can be chemically synthesized (e.g., see

Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N .), large polypeptides derived from CD38/SM38 and the full length CD38/SM38 itself may be

advantageously produced by recombinant DNA technology using techniques well known in the art

for expressing a nucleic acid containing CD38/SM38 gene sequences and/or coding sequences. Such

methods can be used to construct expression vectors containing the CD38/SM38 nucleotide

sequences and appropriate transcriptional and translational control signals. These methods include,

for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic

recombination. (See, for example, the techniques described in Sambrook et al., 1989, supra, and

Ausubel et al., 1989, supra).

[0063] A variety of host-expression vector systems may be utilized to express the CD38/SM38

nucleotide sequences. Where the CD38/SM38 peptide or polypeptide is expressed as a soluble protein or derivative {e.g., peptides corresponding to the intracellular or extracellular domain) and

is not secreted, the peptide or polypeptide can be recovered from the host cell. Alternatively, where

the CD38 peptide or polypeptide is secreted the peptide or polypeptides may be recovered from the

culture media. However, the expression systems also include engineered host cells that express

CD38/SM38 or functional equivalents, anchored in the cell membrane. Purification or enrichment

of the CD38/SM38 from such expression systems can be accomplished using appropriate detergents

and lipid micelles and methods well known to those skilled in the art. Such engineered host cells

themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the CD38/SM38 , but to assess biological activity, i.e., in drug screening

assays. [0064] The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors containing CD38/SM38 nucleotide sequences; yeast transformed

with recombinant yeast expression vectors containing CD38/SM38 nucleotide sequences or mammalian, helminth or insect cell systems harboring recombinant expression constructs containing promoters derived from the genome of mammalian, helminth or insect cells or from mammalian or insect viruses.

[0065] Appropriate expression systems can be chosen to ensure that the correct modification, processing and sub-cellular localization of the CD38/SM38 protein occurs. To tins end, eukaryotic host cells which possess the ability to properly modify and process the CD38/SM38 protein are preferred. For long-term, high yield production of recombinant CD38/SM38 protein, such as that desired for development of cell lines for screening purposes, stable expression is preferred. Rather than using expression vectors which contain origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements and a selectable marker gene, i.e., ik, hgprt, dhfr, neo, and hygro gene, to name a few. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in enriched media, and then switched to a selective media. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that modulate the endogenous activity of the CD38/SM38 gene products. 5.2.2. NON-CELL BASED ASSAYS

[0066] In accordance with the invention, non-cell based assay systems may be used to identify

compounds that interact with, i.e., bind to CD38, and regulate the enzymatic activity of CD38. Such

compounds may act as antagonists or agonists of CD38 enzyme activity and may be used to regulate

cell migration including but not limited to hematopoietically derived cells. Additionally, such

compounds maybe used to regulate the growth, muscle contractility, differentiation, maturation and

reproduction of pathogenic micro-organisms expressing SM38 or structurally related homologues.

Recombinant CD38/SM38 , including peptides corresponding to different functional domains or

CD38/SM38 fusion proteins maybe expressed and used in assays to identify compounds that interact

with CD38/SM38 .

[0067] To this end, soluble CD38/SM38 may be recombinantly expressed and utilized in non-cell based assays to identify compounds that bind to CD38/SM38. Recombinantly expressed

CD38/SM38 polypeptides or fusion proteins containing one or more of the CD38/SM38 functional

domains may be prepared as described above, and used in the non-cell based screening assays. For

example, the full length CD38/SM38 , or a soluble truncated CD38/SM38 , e.g., in which the one

or more of the cytoplasmic and transmembrane domains is deleted from the molecule, a peptide

corresponding to the extracellular domain, or a fusion protein containing the CD38/SM38

extracellular domain fused to a protein or polypeptide that affords advantages in the assay system

(e.g.. labeling, isolation of the resulting complex, etc.) can be utilized. Where compounds that

interact with the cytoplasmic domain are sought to be identified, peptides corresponding to the CD38 cytoplasmic domain and fusion proteins containing the CD38 cytoplasmic domain can be used.

[0068] The CD38/SM38 protein may also be one which has been fully or partially isolated from cell membranes or from the cytosol of cells, or which may be present as part of a crude or semi- purified extract. As a non-limiting example, the CD38 protein may be present in a preparation of cell membranes and the SM38 protein may be present in a preparation of cell cytosol. In particular embodiments of the invention, such cell membranes may be prepared using methods known to those

of skill in the art..

[0069] The principle of the assays used to identify compounds that bind to CD38/SM38 involves preparing a reaction mixture of the CD38/SM38 and the test compound under conditions and for time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. The identity of the bound test compound is then determined.

[0070] The screening assays are accomplished by any of a variety of commonly known methods.

For example, one method to conduct such an assay involves anchoring the CD38/SM38 protein, polypeptide, peptide, fusion protein or the test substance onto a solid phase and detecting CD38/test compound or SM38/test compond complexes anchored on the solid phase at the end of the reaction. i one embodiment of such a method, the CD38/SM38 reactant is anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

[0071] i practice, microtitre plates conveniently can be utilized as the solid phase. The anchored component is immobilized by non-covalent or covalent attachments. The surfaces may be prepared

in advance and stored, h order to conduct the assay, the non-immobilized component is added to

the coated surfaces containing the anchored component. After the reaction is completed, unreacted

components are removed {e.g., by washing) under conditions such that any complexes formed will

remain immobilized on the solid surface. The detection of complexes anchored on the solid surface

can be accomplished in a number of ways. Where the previously non-immobilized component is

pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.

Where the previously non-immobilized component is not pre-labeled, an indirect label can be used

to detect complexes anchored on the solid surface; e.g., using a labeled antibody specific for the

previously non-immobilized component.

[0072] Alternatively, a reaction is conducted in a liquid phase, the reaction products separated

from unreacted components using an immobilized antibody specific for CD38/SM38 protein, fusion

protein or the test compound, and complexes detected using a labeled antibody specific for the other

component of the possible complex to detect anchored complexes.

[0073] In accordance with the invention, non-cell based assays may also be used to screen for

compounds that directly inhibit or activate enzymatic activities associated with CD38/SM38 . Such

activities include but are not limited to ADP-ribosyl cyclase activity, transglycosidation activity, and

NAD+ glycohydrolase activity. To this end, a reaction mixture of CD38/SM38 and a test

compound is prepared in the presence of substrate and the enzymatic activity of CD38/SM38 is

compared to the activity observed in the absence of test compound. Substrates that may be used in the assays for detection of CD38/SM38 enzyme activity include but are not limited to NAD+ and

NADP and labeled forms thereof. Additionally, derivatives of NAD such as Nicotinamide guanine

dinucleotide (NGD) and Nicotinamide 1, N⁶-etheno-adenine dinucleotide (1,N⁶ etheno-NAD) may

be used.

[0074] hi non-limiting embodiments of the invention, a reaction mixture of CD38/SM38 , a test

compound and substrate is prepared and the activity of CD38/SM38 is compared to the activity

observed in the absence of the test compound wherein decrease in the level of CD38/SM38 enzyme

activity in the presence of the test compound indicates that a CD38/SM38 antagonist has been

identified. Alternatively, a reaction mixture of CD38/SM38 , a test compound and substrate is

prepared and the activity of CD38/SM38 is compared to the activity observed in the absence of the test compound wherein an increase in the level of CD38/SM38 enzyme activity in the presence of

the test compound indicates that a CD38/SM38 agonist has been identified.

[0075] The enzymatic activity of CD38/SM38 may be detected in a variety of different ways. For

example, levels of cyclic adenosine diphosphate ribose (cADPR) adenosine diphosphate ribose

(ADPR) and/or nicotinic acid adenine dinucleotide phosphate (NAADP) can be measured using high

performance liquid chromatography (HPLC) or thin layer chromatography (TLC) (Aarhus R et al.,

1995, J. Biochem. Chem. 270:30327-30333; Muller-Steff er HM, J. Biol. Chem. 271:23967-23972;

and Lund FE et al., 1999, J. Immunology 162:2693-2702; Higashida, H. et al., 1997, J. Biol. Chem.

272:3127-3177) in conjunction with the use of radio-labeled substrates such as NAD or NADP or

NA. Additionally, radioimmunoassays (Takahashi K et al., 1995, FEBSLett 371:204-208; Vu CQ et al., 1997, Biochem Biophys Res Commun 236:723-726; Vu et al., Adv Exp Med Biol 419:381-388;

and Graeff RM et al., 1997, Methods Enzymol 280:230-241), bioassays (Aarhus R et al., 1995, J.

Biol Chem. 270:30327-30333; Clapper DL et al, J. Biol. Chem. 262:9561-9568; and Lee et al., J.

Biol. Chem. 264:1608-1615) and/or fluorescent assays (Graeff RM et al., 1996, Biochem. 35:379-

386; Graeff et al, 1994, J. Biol. Chem. 269:30260-30267; and Gadangi P et al., 1996, J. Immunol.

156:1937-1941) may be used for measuring cADPR, ADPR or NAADP levels. In yet another

embodiment of the invention, derivatives of NAD such as NGD (Nicotinamide guanine dinucleotide)

and Nicotinamide 1, N⁶-etheno-adenine dinucleotide (1,N⁶ etheno-NAD) may be used to measure

CD38/SM38 enzyme activity. When the 1,N⁶ etheno-NAD is hydrolysed by CD38, one of the

resulting products will fluoresce (MuUer et al, 1983, Biochem. J. 212:459-464; and Cockayne D et

al., 1998, Blood 92:1324-1333). When the analog NGD is cyclized through the ADP-ribosyl activity

of CD38/SM38 the product forms a fluorescent compound that can be detected by fluorimeter (Graeff et al., 1996, Biochem 35:379-386; and Graeff et al., 1994, J. Biol. Chem. 269:30260-30267).

[0076] h another embodiment of the invention, computer modeling and searching technologies

will permit identification of potential modulators of CD38/SM38 enzyme activity. For example,

based on the knowledge of the Aplysia cyclase active site (Munshi C. et al, 199, J. Biol. Chem. 274:

30770-30777) and the CD38 active site (Lund FE et al, 1999, J. Immunology 162:2693-2702;

Munshi, C et al., 2000, J. Biol. Chem. 275:21566-21571; Graeff R et al., 2001, j. Biol. Chem.

276:12169-12173) and the study of complexes between CD38/SM38 substrates and substrate

anologs, potential modulators of CD38/SM38 activity may be identified. [0077] The three dimensional geometric structure of the active site may be determined using

known methods, including x-ray crystallography, which can determine a complete molecular

structure (see, for example, Prasad GS et al, Nature Struc. Biol. 3:957-964 which describes the

crystal structure of Aplysia ADP ribosyl cyclase). On the other hand, solid or liquid phase NMR can

be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain the partial or complete geometric structure of the CD38 active

site.

[0078] Having determined the structure of the CD38/SM38 active site, candidate modulating

compounds can be identified by searching databases containing compounds along with information

on their molecular structure. Such a search seeks compounds having structures that match the

determined active site structure and that interact with the groups defining the active site. Such a

search can be manual, but is preferably computer assisted. These compounds found from this search

are potential CD38 modulating compounds.

[0079] Alternatively, these methods can be used to identify improved modulating compounds from

an already known modulating compounds. For example, a number of compounds that modulate the

enzyme activity of other enzymes that utilize NAD/NADP as substrates {i.e., PARP family

homologues) have already been identified. The composition of the known compound can be

modified and the structural effects of modification can be determined using experimental and

computer modeling methods applied to the new composition. The altered structure is then compared

to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or substrates of improved specificity or activity.

5. 2. 3.CELL BASED ASSAYS [0080] hi accordance with the invention, a cell based assay system can be used to screen for compounds that modulate the activity of CD38/SM38. In accordance with the invention, a cell-based assay system can be used to screen for compounds that modulate the activity of CD38 and thereby, modulate the chemoattractant induced Ca²⁺ influx and the migration of cells. Additionally, this cell based system can be used to screen for compounds that modulate the activity of SM38, and thereby, modulate intracellular calcium release and/or muscle contractility in cells. To this end, cells that endogenously express CD38/SM38 can be used to screen for compounds. Such cells include, for example, neutrophils, lymphocytes, eosinophils, macrophages and dendritic cells, h addition, S.

mansoni cells that express SM38, may be used to screen for compounds. Alternatively, cell lines, such as 293 cells, COS cells, CHO cells, fibroblasts, and the like, genetically engineered to express CD38/SM38 can be used for screening purposes. For screens utilizing host cells geneticlaly engineered to express a functional CD38 protein, it would be preferred to use host cells that are capable of responding to chemoattractants or inflammatory stimuli. For screens utilizing host cells geneticlaly engineered to exprss SM38, it would be preferable to use cells of S. mansoni origin that are capable of responding to a variety fo stimuli such as acetylcholine or high concentrations of K+ to induce muscle contraction. Further, ooyctes or liposomes engineered to express the CD38/SM38 protein may be used in assays developed to identify modulators of CD38/SM38 activity.

[0081] The present invention provides methods for identifying compounds that alter one of more of the enzymatic activities of CD38/SM38 , including but not limited to, NAD glycohydrolase activity, ADP-ribosyl cyclase activity and/or transglycosidation (exchange) activity. Specifically,

compounds may be identified that promote CD38/SM38 enzyme activities, i.e., agonists, or compounds that inhibit CD38/SM38 enzyme activities, i.e., antagonists. Compounds that inhibit CD38 enzyme activities will be inhibitory for chemoattractant induced calcium responses and cell migration (Figure 2). Compounds that activate CD38 enzyme activity will enhance chemoattractant induced calcium responses and cell migration. Compounds that either activate or inhibit SM38 enzyme activities will alter the viability or functional activities of pathogenic organisms expressing SM38. Such compounds may be compounds that interact with the active site of CD38/SM38 thereby modulating enzyme activity, or compounds that compete/facilitate substrate binding to CD38/SM38 or compete/inhibit catalysis of substrate (Figure 2). Alternatively, compounds may be identified that

modulate the activity of proteins that modify the CD38/SM38 protein, i.e., phosphorylate, ribosylate, etc., and thereby regulate the activity of CD38 (Figure 3). Such proteins include for example, ADP- ribosyl transferases which ribosylate CD38/SM38 and render CD38/SM38 enzymatically inactive, h addition, compounds maybe identified that regulate CD38/SM38 expression and thereby regulate the level of enzyme activity within a cell (Figure 4).

[0082] The present invention provides for methods for identifying a compound that activates

CD38/SM38 enzyme activity comprising (i) contacting a cell expressing CD38/SM38 and chemoattractant receptors with a test compound in the presence of substrate and measuring the level of CD38/SM38 activity; (ii) in a separate experiment, contacting a cell expressing CD38/SM38 protein and chemoattractant receptors with a vehicle control in the presence of substrate and measuring the level of CD38/SM38 activity where the conditions are essentially the same as in part (i), and then (iii) comparing the level of CD38/SM38 activity measured in part (i) with the level of CD38/SM38 activity in part (ii), wherein an increased level of CD38/SM38 activity in the presence of the test compound indicates that the test compound is a CD38/SM38 activator.

[0083] The present invention also provides for methods for identifying a compound that inhibits CD38/SM38 enzyme activity comprising (i) contacting a cell expressing CD38/SM38 and chemoattractant receptors with a test compound in the presence of a chemoattractant and substrate and measuring the level of CD38/SM38 activity; (ii) in a separate experiment, contacting a cell expressing CD38/SM38 and chemoattractant receptors with a chemoattractant and substrate and measuring the level of CD38/SM38 activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of CD38/SM38 activity measured in part (i) with the level of CD38/SM38 activity in part (ii), wherein a decrease level of CD38/SM38 activity in the presence of the test compound indicates that the test compound is a CD38/SM38 inhibitor.

[0084] Depending on the assays used to detect CD38/SM38 activity, the methods described above for identifying activators and inhibitors of CD38/SM38 may include the presence or absence of a chemoattractant in steps (i) and (ii). For example, when assaying directly for CD38/SM38 ADP- ribosyl cyclase activity or the production of CD38/SM38 metabolites, the presence of a chemoattractant or the expression of a chemoattractiant receptor on the test cell may not be required.

However, in instances where, for example, chemotaxis or changes in intracellular calcium levels are

measured in CD-38-expressing cells it may be necessary to include chemoattractants. Alternatively,

when muscle contractility or changes in intracellular calcium levels are measured in SM38-

expressing cells, it may be necessary to include stimulants to activate muscle contraction and/or

calcium release including, but not limited to, acetylcholine, serotonin (Day et al., 1994, Paristol.

108:425-432), FMRF-amide related peptides (FaRPs) (Day et al., 1994, Paristol. 109:455-459) or

high K+ concentrations in the media (Day et al, 1993, Paristol. 106:471-477). Additionally, it will

be necessary to perform these experiments with host cells that express the receptors specific for the

stimulants utilized. Those skilled in the art will be able to determine operative and optimal assay

conditions by employing routine experimentation.

[0085] A "chemoattractant", as defined herein, is a compound or molecular complex that induces

the migration of cells via a mechanism that is dependent on the production of cADPR by CD38.

An example of such a chemoattractant includes, but is not limited to, fMet-leu-Phe (fMLP). Other

chemoattractants that may be used include, eotaxin, GRO-1, IP- 10, SDF-1, BLC, Rantes, MDMA,

MCP-3, MIP3a, IL-8, CLS, ELC, Lymphotactin, PAF, Ltb4, complement c5a and histamine.

[0086] In utilizing the cell systems described above, such cell systems, the cells expressing the

CD38/SM38 protein are exposed to a test compound or to vehicle controls (e.g. , placebos). After

exposure, the cells can be assayed to measure the activity of CD38/SM38 or the activity of the CD38

dependent signal transduction pathway itself can be assayed. [0087] The ability of a test molecule to modulate the activity of CD38/SM38 may be measured

using standard biochemical and physiological techniques. Responses such as activation or

suppression of CD38/SM38 ADP-ribosyl cyclase activity or the production of CD38/SM38

metabolites such as cADPR and/or NAADP can be measured. Levels of cADPR, ADPR and/or

NAADP can be measured using HPLC or TLC in conjunction with the use of radio-labeled

substrates such as NAD or NADP or NA. Additionally, radioimmunoassays, bioassays and/or

fluorescent assays, such as those discussed in Section 5.1.1, supra, may be used for measuring

cADPR or NAADP levels. In yet another embodiment of the invention, derivatives of NAD such

as NGD (Nicotinamide guanine dinucleotide) and Nicotinamide 1, N⁶-etheno-adenine dinucleotide

(1,N⁶ etheno-NAD) maybe used to measure CD38/SM38 activity.

[0088] Test compounds may also be assayed utilizing cell based calcium and/or migration assays

to identify compounds that are capable of inhibiting or activating chemoattractant induced CD38

dependent calcium responses and cell migration. In non-limiting embodiments of the invention,

changes in intracellular Ca²⁺ levels may be monitored by the fluorescence of Ca²⁺ indicator dyes

such as frido, Fluo-3 and Fura-Red, etc. Further, changes in membrane potential resulting from

modulation of the CD38/SM38 enzyme activity can be measured using a voltage clamp or patch

recording methods. Directed migration of cells may also be monitored by standard chemotaxis

assays in modified Boyden chambers or on slides. Such assay systems are described in further detail

in the working example of the present specification (See, Example 6). Muscle contractility may also

be measured by standard assays described in detail in the literature (for example: (Day et al, 1994 Parasitology 109:455-9) and references therein).

[0089] After exposure to the test compound, or in the presence of a test compound, cells can be

stimulated with a chemoattractant such as fMLP or a muscle activator such as high K+

concentrations, and changes in intracellular calcium levels, cADPR or NAADP levels, muscle

contractility and/or cell migration may be measured. These measurements will be compared to cells

treated with the vehicle control. Increased levels of intracellular Ca²⁺, increased production of

cADPR, increases in muscle contractility and/or increased migration of cells toward a

chemoattractant in the presence of a test compound indicates that the compound acts as an agonists

to increase the Ca2+ response increase muscle contractility and increase chemoattractant induced

CD38 dependent cell migration. Decreased levels of intracellular Ca2+, decreased production of

cADPR, decreased muscle contractility and/or decreased migration of cells toward a chemoattractant

in the presence of a test compound indicates that the compound acts as an antagonist and inhibits the

Ca2+ response, decreases muscle contractility and inhibits chemoattractant induced CD38 dependent

cell migration (see, for example, Figures 2 and 3).

[0090] In addition, the assays of the invention may be used to identify compounds that (i) function as substrates of CD38/SM38 enzymatic activity and are converted into agonists or antagonists of

cADPR dependent Ca2+ signal transduction pathway (Figure 5). A compond fitting these

specifications is described in further detail in the working exmaple of the present specification

(Example 6, Figure 11). Alternatively, the assays of the invention may be used (ii) to identify

compounds that specifically interfere with the cADPR mediated Ca2+ signal transduction pathways (Figure 6). In a non-limiting embodiment of the invention, test compounds may include chemical

derivatives of any known and unknown substrates of CD38/SM38 (for example, the substrate analog

8-Br-βNAD is converted into the modified product 8-Br-cADPR which acts as an antagonist of

cADPR mediated Ca2+ signal transduction). The test substrate may be administered to cells

expressing CD38/SM38 and the appropriate chemoattractant receptors in the presence of the

chemoattractant or muscle stimulant. Conversion of the modified test substrate into a modified

product that is capable of modulating the activity of cADPR can be measured utilizing the methods

described above. Test substrates may also be assayed to determine their effect on calcium influx,

muscle contractility and/or cell migration. Intracellular Ca2+ accumulation and directed migration

to a chemoattractant can be measured in cells treated with the test substrate and the chemoattractant

and compared to cells receiving the non-modified substrate, i.e., NAD and a chemoattractant.

Compounds which are converted into modified products, i.e., 8-Br-cADPR, and competitively or

non-competitively inhibit cADPR induced calcium responses, muscle contractility or directed

migration will be identified as antagonists of the cADPR Ca²⁺ signaling pathway, while compounds

that are converted into modified products that are competitive or non-competitive agonists of the

cADPR Ca²⁺ signaling pathway will be defined as agonists or activators.

[0091] In yet another embodiment of the invention, compounds that directly alter {i.e., activate or

inactivate) the activity of cADPR, i.e., induced calcium release and cell migration, can be tested in

assays. Such agonists or antagonists would be expected to modulate the influx of Ca2+ into the cell

resulting in changes in the cell's migratory activity or ability to contract. Antagonists would have reduced Ca2+ responses, reduced contractility and/or reduced migration in the presence of a

chemoattractant. Examples of antagonists include, but are not limited to 8-NH -cADPR, 8-Br-

cADPR, 8-CH₃-cADPR, 8-OCH₃-cADPR and 7-Deaza-8-Br-cADPR. A compound fitting these

specifications is described in further detail in the working example of the present specification

(Example 6, Figure 10). Agonists would have increased Ca2+ responses, increased contractility

and/or increased migration in the presence of chemoattractants. Examples of agonists include but

are not limited to 2'-deoxy-cADPR, 3'-deoxy-cADPR and 2'-phospho-cADPR. Assays for direct

measurement of cAPDR activity include the bioassays such as those described by Howard et al.

(1995, Science 262:1056); Galione et al. (1993, Nature 365:456-459) and Lee and Aarhus (1991,

Cell Regulation 2:203-209).

[0092] Further, the assays of invention may identify compounds that are capable of activating

CD38/SM38 enzyme activity, i.e., agonists, but which desensitize the calcium pathway by depletion

of intracellular calcium stores. Such desensitization may, in some instances, lead to inhibition of

cell migration or muscle contraction due to the depletion of calcium stores. Thus compounds may

be identified that function as agonists in CD38/SM38 enzyme assays but function as antagonists in

chemotaxis or muscle contraction assays. Such assays and compounds are within the scope of the

present invention.

5.2.4. ASSAY FOR COMPOUNDS THAT REGULATE THE EXPRESSION OF CD38/SM38

[0093] In accordance with the invention, a cell based assay system can be used to screen for compounds that modulate the expression of CD38/SM38 within a cell. Assays may be designed to

screen for compounds that regulate CD38/SM38 expression at either the transcriptional or

translational level. In one embodiment, DNA encoding a reporter molecule can be linked to a

regulatory element of the CD38/SM38 gene and used in appropriate intact cells, cell extracts or

lysates to identify compounds that modulate CD38/SM38 gene expression. Such reporter genes may

include but are not limited to chloramphenicol acetyltransferase (CAT), luciferase, β-glucuronidase

(GUS), growth hormone, or placental alkaline phosphatase (SEAP). Such constructs are introduced

into cells thereby providing a recombinant cell useful for screening assays designed to identify

modulators of CD38/SM38 gene expression.

[0094] Following exposure of the cells to the test compound, the level of reporter gene expression

may be quantitated to determine the test compound's ability to regulate CD38/SM38 expression.

Alkaline phosphatase assays are particularly useful in the practice of the invention as the enzyme is

secreted from the cell. Therefore, tissue culture supernatant may be assayed for secreted alkaline

phosphatase. In addition, alkaline phosphatase activity may be measured by calorimetric,

bioluminescent or chemiluminescent assays such as those described in Bronstein, I. et al. (1994,

Biotechniques 17: 172-177). Such assays provide a simple, sensitive easily automatable detection

system for pharmaceutical screening.

[0095] To identify compounds that regulate CD38/SM38 translation, cells or in vitro cell lysates

containing CD38/SM38 transcripts may be tested for modulation of CD38/SM38 mRNA translation.

To assay for inhibitors of CD38/SM38 translation, test compounds are assayed for their ability to modulate the translation of CD38/SM38 mRNA in in vitro translation extracts.

[0096] hi an embodiment of the invention, the level of CD38/SM38 expression can be modulated

using antisense or ribozyme approaches to inliibit or prevent translation of CD38/SM38 mRNA

transcripts or triple helix approaches to inhibit transcription of the CD38/SM38 gene. Such

approaches may be utilized to treat disorders such as inflamation and allergies where inhibition of

CD38/SM38 expression is designed to prevent hematopoietically-derived cell migration or inhibition

of SM38 is designed to alter S. mansoni physiology and pathogenesis.

[0097] Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are

complementary to CD38/SM38 mRNA. The antisense oligonucleotides will bind to the

complementary mRNA transcripts and prevent translation. Absolute complementarity, although

preferred, is not required. One skilled in the art can ascertain a tolerable degree of mismatch by use

of standard procedures to determine the melting point of the hybridized complex.

[0098] In yet another embodiment of the invention, ribozyme molecules designed to catalytically

cleave CD38/SM38 mRNA transcripts can also be used to prevent translation of CD38/SM38

mRNA and expression of CD38/SM38. (See, e.g., PCT International Publication WO90/11364,

published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225). Alternatively, endogenous CD38/SM38 gene expression can be reduced by targeting deoxyribonucleotide sequences

complementary to the regulatory region of the CD38/SM38 gene {i.e., the CD38 promoter and or

enhancers) to form triple helical structures that prevent transcription of the CD38/SM38 gene in targeted hematopoietically-derived cells in the body. (See generally, Helene, C. et al., 1991, Anticancer Drug Des. 6:569-584 and Maher, LJ, 1992, Bioassays 14:807-815).

[0099] The oligonucleotides of the invention, i.e., antisense, ribozyme and triple helix forming oligonucleotides, may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied

Biosystems, etc.). Alternatively, recombinant expression vectors may be constructed to direct the expression of the oligonucleotides of the invention. Such vectors can be constructed by recombinant DNA technology methods standard in the art. hi a specific embodiment, vectors such as viral vectors may be designed for gene therapy applications where the goal is in vivo expression of inhibitory oligonucleotides in targeted cells.

5.2.5. COMPOUNDS THAT CAN BE SCREENED IN ACCORDANCE WITH THE INVENTION

[0100] The assays described above can identify compounds which modulate CD38/SM38 activity. For example, compounds that affect CD38/SM38 activity include but are not limited to compounds that bind to CD38/SM38 , and either activate enzyme activities (agonists) or block enzyme activities (antagonists). Alternatively, compounds may be identified that do not bind directly to CD38/SM38 but are capable of altering CD38/SM38 enzyme activity by altering the activity of a protein that regulates CD38 enzyme activity (see, Figure 3) Compounds that are substrates of CD38 that are converted into modified products that activate or inhibit the cADPR Ca2+ signal transduction pathway can also be identified by the screens of the invention. Compounds that directly activate or inhibit the cADPR Ca2+ signal transduction pathway in cells

can also be identified. Additionally, compounds that activate CD38/SM38 enzyme activity

resulting in desensitization of the calcium pathway may be identified. Such desensitizing

compounds would be expected to inhibit cell migration. Further, compounds that affect

CD38/SM38 gene activity (by affecting CD38/SM38 gene expression, including molecules, e.g.,

proteins or small organic molecules, that affect transcription or interfere with splicing events so

that expression of the full length or the truncated form of the CD38/SM38 can be modulated) can

be identified using the screens of the invention.

[0101] The compounds which may be screened in accordance with the invention include, but are

not limited to, small organic or inorganic compounds, peptides, antibodies and fragments thereof,

and other organic compounds (e.g., peptidomimetics) that bind to CD38/SM38 and either mimic

the activity triggered by any of the known or unknown substrates of CD38/SM38 {i.e., agonists)

or inhibit the activity triggered by any of the known or unknown substrates of CD38/SM38 {i.e.,

antagonists). Compounds that bind to CD38/SM38 and either enhance CD38/SM38 enzyme

activities {i.e., ADP-ribosyl cyclase activity, NAD glycohydrolase activity, transglycosidation

activity), i.e., agonists, or compounds that inhibit CD38/SM38 enzyme activities, i.e.,

antagonists, in the presence or absence of the chemoattractant or muscle stimulant will be

identified. Compounds that bind to proteins that alter/modulate the enzyme activity of

CD38/SM38 will be identified. Compounds that mimic natural substrates , i.e., NAD(P) and are

converted by CD38/SM38 enzyme activities into products that act as agonists or antagonists of the cADPR induced calcium release pathway can be identified. Compounds that directly activate

or inhibit the cADPR Ca2+ signal transduction pathway in cells can be identified.

[0102] Compounds may include, but are not limited to, peptides such as, for example, soluble

peptides, including but not limited to members of random peptide libraries (see, e_^g., Lam, K.S.

et al., 1991, Nature 354:82-84; Houghten, R. et al., 1991, Nature 354:84-86); and combinatorial

chemistry-derived molecular library made of D- and/or L- configuration amino acids,

phosphopeptides (including, but not limited to, members of random or partially degenerate,

directed phosphopeptide libraries; (see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),

antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,

chimeric or single chain antibodies, and FAb, F(ab') and FAb expression library fragments, and

epitope binding fragments thereof), and small organic or inorganic molecules.

[0103] Other compounds which may be screened in accordance with the invention include but

are not limited to small organic molecules that affect the expression of the CD38/SM38 gene or

some other gene involved in the CD38/SM38 signal transduction pathway (e.g., by interacting

with the regulatory region or transcription factors involved in gene expression); or such

compounds that affect the enzyme activities of the CD38/SM38 or the activity of some other

factor involved in modulating CD38/SM38 enzyme activity, such as for example, a protein that

ribosylates CD38/SM38 and thereby inactivates CD38/SM38 enzyme activities. 5.3. COMPOSITIONS CONTAINING MODULATORS

OF CD38/SM38 AND THEIR USES

[0104] The present invention provides for methods of modulating cell migration comprising

contacting a cell expressing CD38 with an effective amount of a CD38 modulating compound,

such as a CD38 agonist or antagonist identified using the assays as set forth in Section 5.1 supra.

Additionally, the present invention provides for methods of modulating calcium responses and/or

muscle contractility comprising contacting a cell expressing SM38 with an effective amount of a

SM38 modulating compound, such as a SM38 agonist or antagonist identified using the assays as

set forth in Section 5.1 supra. An "effective amount" of the CD38/SM38 inhibitor, i.e., antagonist, is an amount that decreases chemoattractant induced cell migration decreases

intracellular calcium levels, decreases muscle contraction and/or that is associated with a

detectable decrease in CD38/SM38 enzyme activity as measured by one of the above assays. An

"effective amount" of the CD38/SM38 activator, i.e., agonist, is an amount that subjectively

increases chemoattractant induced cell migration, increases intracellular calcium levels, increases

muscle contraction and/or that is associated with a detectable increase in CD38/SM38 enzyme

activity as measured by one of the above assays. Compositions of the invention also include

modified CD38/SM38 substrates, modulators of CD38/SM38 expression and

agonists/antagonists of cADPR.

[0105] The present invention further provides methods of modulating cell migration in a subject,

comprising administering to the subject, a composition comprising a compound that modulates

CD38 enzyme activity identified as set forth in Section 5.1 supra. The composition may comprise an amount of CD38 enzyme activator or inhibitor, modulators of CD38 expression,

modified CD38 substrates, or direct agonists/antagonists of cADPR controlled Ca2+ responses.

Accordingly, the present invention provides for compositions comprising CD38 activators and

inhibitors.

[0106] The present invention provides for compositions comprising an effective amount of a

compound capable of modulating the activity of CD38, the expression of CD38 and/or the

activity of cADPR thereby regulating the migratory activity of cells, and a pharmaceutically

acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means

approved by a regulatory agency of the Federal or a state government or listed in the U.S.

Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more

particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with

which the therapeutic is administered. Examples of suitable pharmaceutical carriers are

described in "Remington's Pharmaceutical sciences" by E.W. Martin.

[0107] The invention provides for treatment or prevention of various diseases and disorders

associated with cell migration by administration of a compound that regulates the expression or

activity of CD38. Such compounds include but are not limited to CD38 antibodies; CD38

antisense nucleic acids, CD38 agonists and antagonists (see, Figures 2-3), modified CD38 substrates (see, Figure 5) and cADPR agonists and antagonists (see, Figure 6). In a non-limiting

embodiment of the invention, disorders associated with hematopoietic derived cell migration are

treated or prevented by administration of a compound that regulates CD38 activity. Such disorders include but are not limited to inflammation, ischemia, asthma, auto-immune disease,

diabetes, allergies, infections, arthritis and organ transplant rejections.

[0108] The compounds of the invention are preferably tested in vitro, and then in vivo for a

desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays

which can be used to determine whether administration of a specific therapeutic is indicated,

include in vitro cell culture assays in which cells expressing CD38 are exposed to or otherwise

administered a therapeutic compound and the effect of such a therapeutic upon CD38 activity is

observed. In a specific embodiment of the invention the ability of a compound to regulate, i.e.,

activate or inhibit cell migration may be assayed.

[0109] The present invention further provides methods of modulating the muscle contraction or

other physiologic parameters in helminths such as S. mansoni by administering to helminth

infected subject, a composition comprising a compound that modulates SM38 enzyme activity

identified as set forth in Section 5.1 supra. The composition may comprise an amount of SM38

enzyme activator or inhibitor, modulators of SM38 expression, modified SM38 substrates, or

direct agonists/antagonists of cADPR controlled Ca2+ responses. Accordingly, the present

invention provides for compositions comprising SM38 activators and inhibitors.

[0110] The present invention provides for compositions comprising an effective amount of a

compound capable of modulating the activity of SM38, the expression of SM38 and/or the activity of c ADPR thereby regulating the activity and viability of the parasite, and a

pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically

acceptable" means approved by a regulatory agency of the Federal or a state government or listed

in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and

more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or

vehicle with which the therapeutic is administered. Examples of suitable pharmaceutical carriers

are described in "Remington's Pharmaceutical sciences" by E.W. Martin.

[0111] The invention provides for treatment or prevention of various diseases and disorders

associated with helminth infections. Such compounds include but are not limited to SM38

antibodies; SM38 antisense nucleic acids, SM38 agonists and antagonists (see, Figures 2-3),

modified SM38 substrates (see, Figure 5) and cADPR agonists and antagonists (see, Figure 6).

In a non-limiting embodiment of the invention, disorders associated with helminth infection are

treated or prevented by administration of a compound that regulates SM38 activity. Such

disorders include but are not limited to granuloma formation and fibrosis in the liver and lung.

[0112] The compounds of the invention are preferably tested in vitro, and then in vivo for a

desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays

which can be used to determine whether administration of a specific therapeutic is indicated,

include in vitro cell culture assays in which cells expressing SM38 are exposed to or otherwise

administered a therapeutic compound and the effect of such a therapeutic upon SM38 activity is observed. In a specific embodiment of the invention the ability of a compound to regulate, i.e., activate or inhibit muscle contractility or intracellular calcium accumulation. Additionally, the

compounds of the invention may be assayed for their effect on S. mansoni pathogenesis, growth,

differentiation, and reproduction in a mouse model for S. mansoni infection. Such assays would

include the testing for effects on proliferation of parasites, maturation of female worms, quantity

of granulomas in liver and lung, quantity of eggs in liver, lung bladder and intestines , quatity of

worms in lung and liver and quantity of miracidia detected in urine and feces.

[0113] Additionally, the compounds of the invention may be assayed for their effect on S.

mansoni. pathogenesis, growth, differentiation, and reproduction. Such compounds could be

tested in a mouse model for S. mansoni infection. Such assays would include the testing for

effects on proliferation of parasites, quantity of granulomas in liver and lung, quantity of eggs in

liver, lung bladder and intestines and quantity of miracidia detected in urine and feces.

[0114] The invention provides methods of treatment and/or prophylaxis by administration to a

subject of an effective amount of a compound of the invention. In a preferred aspect, the

compound is substantially purified. The subject is preferably an animal, and is preferably a

mammal, and most preferably human.

[0115] Various delivery systems are known and can be used to administer a compound capable

of regulating CD38 activity, cADPR, or CD38 expression, e.g., encapsulation in liposomes,

microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-

mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal,

intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be

administered by any convenient route, for example by infusion or bolus injection, by absorption

through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)

and may be administered together with other biologically active agents. Administration can be

systemic or local. Pulmonary administration can also be employed, e.g., by use of an inhaler or

nebulizer, and formulation with an aerosolizing agent.

[0116] In a specific embodiment, it maybe desirable to administer the compositions of the

invention locally to a specific area of the body; this may be achieved by, for example, and not by

way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a

wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or

by means of an implant, said implant being of a porous, non-porous, or gelatinous material,

including membranes, such as sialastic membranes, or fibers.

[0117] The present invention also provides pharmaceutical compositions. Such compositions

comprise a therapeutically effective amount of a compound capable of regulating CD38 activity,

cADPR activity or CD38 expression and a pharmaceutically acceptable carrier. In a specific

embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of

the Federal or a state government or listed in the U.S. Pharmacopeia or other Generally

recognized pharmacopeia for use in animals, and more particularly in humans. The term

"carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of

mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The

formulation should suit the mode of administration.

[0118] The amount of the compound of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose response curves derived from in vitro or animal model test systems. Additionally, the administration of the compound could be

combined with other known efficacious drugs if the in vitro and in vivo studies indicate a

synergistic or additive therapeutic effect when administered in combination.

[0119] The invention also provides a pharmaceutical pack or kit comprising one or more

containers filled with one or more of the ingredients of the pharmaceutical compositions of the

invention, optionally associated with such container(s) can be a notice in the form prescribed by a

governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological

products, which notice reflects approval by the agency of manufacture, use or sale for human

administration.

6. EXAMPLE: NEUTROPHILS REQUIRE CD38 FOR CHEMOTAXIS, CAPACITATIVE Ca+ ENTRY AND BACTERIAL CLEARANCE

[0120] The subsection below describes data demonstrating that calcium entry in chemoattractant

activated neutrophils is controlled by cADPR, a product of the CD38 enzyme reaction. The

capacitative calcium influx, controlled by the cADPR produced by CD38, is required for

neutrophils to migrate efficiently to chemoattractants.

6.1. MATERIALS AND METHODS

6.1.1. MICE

[0121] C57BL/6J x 129 CD38KO F2 animals(Cockayne et al., 1998 Blood 92:1324-1333) were backcrossed 6 generations (N6) to C57BL/6J and then inbred to produce homozygous congenic C57BL/6J.129 CD38KO mice. CD38-Rag-2 double KO (dKO) mice were produced by crossing

C57BL/6J.129 (N6) CD38KO mice with C57BL/6J.129 (N8) Rag-2 KO mice (Shin Kai et al., 1992 Cell 68:855-867) and then mating the offspring to obtain homozygous double KO animals.

Bone marrow chimeric mice were produced by transplanting lxlO⁷ whole bone marrow cells

isolated from WT or CD38KO mice into lethally irradiated (950 rad) WT hosts. All mice were

bred and maintained in the Trudeau Institute Animal Breeding Facility.

6.1.2. cADPR CONTENT MEASUREMENTS.

[0122] Mouse tissues were isolated from whole-body perfused WT or CD38KO mice and were

flash frozen in liquid nitrogen. Bone marrow myeloid cells were flushed from the tibias and

femurs of Rag-2KO or Rag-2-CD38 dKO mice. cADPR content in mouse tissues and bone

marrow myeloid cells was then measured as previously described (Vu et al., 1997 Biochem

Biophys Res Commun 236:723-726).

[0123] S. pneumoniae infection. Mice were infected intra-tracheally with 100 or 1000 CFU S.

pneumoniae type 4 (Klein Strain) from American Type Tissue Culture (Rockville, Maryland).

Blood, bronchial-aveolar lavage fluid (BAL) and lung tissue were collected from infected mice

(Garvy et al., 1996 Inflammation 20:499-512). Bacterial titers in lung homogenate and blood

were calculated on a per lung basis or per ml of blood. BAL cells were enumerated from cyto-

centrifuge preparations. 6.1.3. IN VITRO CHEMOTAXIS ASSAYS

[0124] Bone marrow neutrophils were purified (95% purity) by positive selection using

biotinylated GR-1 (PharMingen) and MACS Sfreptavidin Microbeads (Miltenyi Biotec, Auburn

CA). Chemotaxis assays(Falk et al., 1980 J. Immuno. Methods 33:239-247) were performed

using 24-well transwell plates with a 3 μm pore size polycarbonate filter (Costar, Cambridge,

MA). Medium (HBSS+Ca2⁺+Mg2⁺), fMLP (1 μM, Sigma, St. Louis, MO), or IL-8 (100 nM,

Sigma) was placed in the lower and/or upper chamber in a checkerboard format. lxlO⁵

neutrophils were loaded in the upper chamber and the plates were incubated at 37° C for 45 min.

The transmigrated cells were collected from the lower chamber, fixed and counted on the flow

cytometer (FACS Calibur, Becton Dickinson, San Jose CA). To determine the absolute number

of cells in each sample, a standard number of 20 μM size fluorescent microspheres

(Polysciences, Inc. Warrington, PA) was added to each tube and counted along with the cells.

The total number of transmigrated cells = the number of counted neutrophils X total number of

beads/beads counted. In some experiments, neutrophils were incubated in EGTA (2mM) or pre¬

heated for 20 min with 8-Br-cADPR (25-100 μM, Sigma) or N(8-Br-A)D+ (1.0 mM).

6.1.4. CD38 EXPRESSION

[0125] Bone marrow, blood or peritoneal cavity cells were isolated from WT or CD38KO mice

and stained with anti-mouse GR-1 FITC, anti-mouse MAC-1 PE and anti-mouse CD38 APC (PharMingen, San Diego CA). Human peripheral blood neutrophils were isolated on a ficoll

gradient and then stained with anti-hCD15-FITC (Becton Dickinson, San Jose CA) and anti- hCD38-Biotin (Caltag Laboratories, Burlingame CA). Mouse and human neutrophils were

analyzed by flow cytometry, gating on the MAC-1⁺GR-1⁺ for mouse neutrophils and CD15⁺ for

human neutrophils. To induce an inflammatory response, mice were injected with 1 ml 3%

thioglycollate medium intra-peritoneally (Becton Dickinson, Cockeysville MD). The animals

were sacrificed 12 hrs post-injection, and the cells infiltrating the peritoneal cavity were

collected.

6.1.4. MEASUREMENT OF CD38 CYCLASE ACTIVITY

[0126] Measurement of CD38 cyclase activity. lxlO⁶ purified bone marrow neutrophils were

incubated for 20 min at 37° C in 100 μl HBSS in a 96 well microplate. NGD (40 μM) (Sigma)

was added and the enzymatic conversion of NGD⁺ to cGDPR was measured fluorometrically

(Graeff et al., 1994 J. biol. Chem. 269:30260-30267) over the next 10 minutes (415 nm emission

and 300 nm excitation).

6.1.5. RYR-3 MRNA EXPRESSION IN NEUTROPHILS

[0127] cDNA was prepared from RNA isolated from purified bone marrow neutrophils or brain

tissue. 30 cycles (annealing temperature 61°C) RT-PCR was performed with 0.03-2 μg input

cDNA and RyR-3 specific primers (Guse et al., 1999 Nature 398:70-73).

[0128] Synthesis of N(8-Br-A)D⁺. N(8-Br-A)D⁺ was synthesized as previously described (Abdallah et al. 1975 Eur. J Biochem 50:475-481). 6.1.6. INTRACELLULAR CALCIUM MEASUREMENTS

[0129] Purified bone marrow neutrophils were resuspended in cell loading media (HBSS with

Ca2⁺ and Mg2⁺ + 1 % FBS + 4 mM probenecid) at lxl 0⁷ cells/ml. The cells were incubated at

37° C for 30 min with the fluorescent dyes Fluo-3 AM (4 μg/ml) and Fura Red AM (10 μg/ml)

(Molecular Probes, Eugene OR) and then washed twice and resuspended in cell loading medium

or calcium-free medium at lxlO⁶ cells/ml. In some experiments, cells were permeabilized in 5

μM digitonin in calcium-free media. In other experiments, cells were preincubated with EGTA

(2mM), 8-Br-cADPR (10-100 μM), ruthenium red (Sigma) or N(8-Br-A)D⁺ (ImM) and then

stimulated with the carrier control (DMSO 0.01%), fMLP (1 μM), IL-8 (100 nM), ryanodine (1

μM), cADPR (100 μM) or thapsigargin (1 μM). The accumulation of [Ca²+]i in individual cells

was assessed by flow cytometry measuring the fluorescence emission of Fluo-3 in the FL-1

channel and Fura-Red in the FL-3 channel. Data was analyzed using FlowJo 3.2 (Tree Star, Inc.

San Carlos, CA). The relative [Ca2⁺]i was expressed as the ratio between Fluo-3 and Fura Red

mean fluorescence intensity over time.

6.2. RESULTS

[0130] CD38 is the primary ADP-ribosyl cyclase expressed in lymphoid tissues. To directly test

the requirement for CD38 and cADPR in calcium-sensitive immunologic responses in vivo,

CD38 knockout (CD38KO) mice where generated (Cockkayne et al. 1998 Blood 92:1324-1333).

To determine whether CD38 is the primary cyclase expressed in mice, the cADPR content in

tissues and cells isolated from CD38KO and C57BL/6J wild-type (WT) mice were compared(Table 1).

Table 1. Comparison of cADPR content in tissues isolated from CD38KO and WT animals.

Tissue cADPR content WT tissue cADPR content CD38K0

(pmol/mg protein) tissue

(pmol/mg protein)

Spleen 2.108 ± 0.334 0.298 ± 0.091*

Thymus 0.769 ± 0.182 0.335 ± 0.088**

BM myeloid 0.633 ± 0.111 0.257 ± 0.032*

Lung 0.847 ± 0.213 0.480 ± 0.069

Kidney 0.488 ± 0.119 0.418 ± 0.070

Heart 1.249 ± 0.324 1.014 ± 0.237

Brain 3.865 ± 0.866 3.127 + 0.316

Extracts were prepared from tissues isolated from 8-12 wk old CD38KO or WT mice or from bone marrow (BM) myeloid cells isolated fromRag-2KO or Rag-2-CD38 double KO mice and were analyzed for cADPR content. Three separate purifications and analyses were performed on tissues isolated from 3 mice/analysis. *P=0.01, ** =0.07; Anova analysis. Limit of detection, 0.2 pmol/mg protein.

[0131] WT tissues containing primarily lymphoid or myeloid cells, such as spleen, thymus and

lymphoid deficient bone marrow (myeloid cells), had easily detectable levels of cADPR. In

contrast, cADPR was not detected in lymphoid or myeloid tissues isolated from CD38KO mice.

However, the cADPR content of CD38KO tissues such as brain, kidney and heart was nearly

equivalent to the cADPR content of the same WT tissues. Thus, other unknown cyclases must be

responsible for cADPR production in organs such as brain and heart, however, CD38 is the

predominant ADP-ribosyl-cyclase expressed by myeloid and lymphoid cells.

[0132] CD38 deficient mice are more susceptible to bacterial infection. To test the requirement

for CD38 and cADPR in innate inflammatory immune responses, CD38KO and WT mice were

infected with Streptococcus pneumoniae and assessed survival (Figure 7A). It was observed that the LD50 for CD38 KO animals is at least 10-fold lower than for WT mice, as 100 colony

forming units (CFU) killed 50% of the CD38KO mice within 2.5 days of infection, while 1000

CFU were required to kill 50% of the WT animals in the same time period.

[0133] Since CD38 is expressed by the responding immunocytes and the bronchial epithelium

(Fernandez JE et al., J. biol Reg Homeost Agents 12:81-91), WT or CD38KO bone marrow was

transplanted into irradiated WT hosts to test whether CD38 expression in the lung and/or

immune system was necessary for protection. The reconstituted chimeric animals possessed

either CD38+ or CD38 -deficient bone-marrow derived cells, while all other cell types, including

the bronchial epithelium, were of WT origin in both groups of animals. The reconstituted mice

were then infected with S. pneumoniae and survival was monitored (Figure 7B). Reconstituted

animals receiving CD38KO bone marrow were much more susceptible to infection compared to

mice receiving WT bone marrow, indicating that the increased susceptibility of CD38KO mice to

S. pneumoniae infection is due to the loss of CD38 on bone marrow-derived lymphoid and/or

myeloid cells.

[0134] To determine whether the increased susceptibility of CD38KO animals to S. pneumoniae

was due to an inability to restrain bacterial growth and spreading to systemic sites, CD38KO and

WT mice were infected with 1000 CFU of S. pneumoniae and bacterial titers were assessed in

lung and blood 12 hours post-infection (Figure 7C). The bacterial titer in the lungs of CD38KO

mice was increased five-fold compared to WT controls. However, the bacterial burden in the

blood of the CD38KO mice was 200-500 times greater than in WT mice, indicating that the bacteria rapidly disseminate in CD38KO mice.

[0135] To determine whether myeloid or lymphoid cells were responsible for the increased

bacterial spreading, Rag-2 KO mice (Shin Kai, et al., 1992 Cell 68:855-867) (which lack

lymphocytes but can express CD38 on all myeloid cells) and CD38-Rag-2 double knockout mice

(which lack lymphocytes and cannot express CD38 on their myeloid cells) were infected with

1000 CFU S. pneumoniae and then bacterial titers were determined in lung and blood 12 hours

later (Figure 7C). The bacterial titers in the lungs and blood of the lymphoid-deficient CD38-

Rag-2 double KO mice were as high as those seen in the CD38KO mice and were significantly

increased when compared to Rag-2 KO or WT mice. Thus, CD38 deficient myeloid cells are

responsible for the increased susceptibility of CD38KO mice to S. pneumoniae.

[0136] CD38 deficient neutrophils do not accumulate at sites of infection and inflammation. To

test whether myeloid cells were appropriately recruited to the lungs of S. pneumoniae-mfected

CD38KO animals, CD38KO and WT mice were infected and then the cells that were recruited to

the lung airways after infection were enumerated. The total number of cells in the airways of

CD38KO and WT animals increased equivalently from 6 to 18 hours post-infection (Figure 8 A).

However, neutrophils were the predominant cell type found in the lungs of WT animals 12-18

hours post-infection, while the cellular infiltrate in the lungs of the CD38KO animals was

composed primarily of macrophages (Figure 8B-C). Thus, CD38 appears to be required for sustained recruitment of neutrophils to the site of infection and inflammation. [0137] CD38 deficient neutrophils make a defective chemotactic response to the chemoattractant

fMLP. Neutrophils migrate to sites of infection in response to gradients of chemokines and

chemoattractants that are produced by the local cells and by the invading pathogen (Hub et al.

1996 Chemoattractant Ligands and Their Receptors (ed. Horuk) 301-325 (CRC Press, Boca

Raton, FL); Servant G. et al., 2000 Science 287:1037-1040; Gao, J.L., 1999 J. Exp. Med

189:657-662). Chemoattractants rapidly activate neutrophils and induce random migration

(chemokinesis). If a chemotactic gradient exists, the activated neutrophils polarize their leading

edge toward the highest concentration of the gradient and migrate directionallylό (chemotaxis).

It has been previously demonstrated that neutrophils home to sites of infection upon stimulation

of their N-formylpeptide receptor (FPR) by bacterially-derived formylated peptides such as

formyl-methionyl-leucyl-phenyalanine (fMLP). To test whether CD38KO neutrophils were defective in their ability to chemotax to fMLP, the ability of CD38KO and WT neutrophils to

migrate by chemokinesis and chemotaxis in a transwell checkerboard assay was determined (Falk

et al., 1980 J. Immunol. Methods 33:239-247) (Figure 8D). When fMLP was absent from the top

and bottom chamber, or when fMLP was placed only in the top chamber, few (<2300 cells), but

equivalent numbers, of the CD38KO and WT neutrophils migrated to the bottom chamber.

When an equal concentration of fMLP was present in the top and bottom chamber (chemokinesis

conditions), increased, but similar, numbers of WT and CD38KO neutrophils migrated to the

bottom chamber, indicating that activation-induced chemokinesis to fMLP was equivalent

between CD38KO and WT neutrophils. When fMLP was present in the bottom chamber only (chemotaxis conditions), the migration of WT neutrophils to the bottom chamber was further increased. However, CD38KO neutrophils migrated only marginally better in the presence of a chemotactic gradient than in the absence of a fMLP gradient, indicating that CD38KO neutrophils can be activated to migrate by bacterial chemoattractants but are unable to follow the chemotactic gradient. To determine if this was a general property of CD38KO neutrophils, the same experiments were performed using the chemokine IL-8, which is a potent activator of

neutrophils (Baggiolini et al., 1989 J. Clin. Invest 84:1045-1049). hi contrast to what was observed with fMLP, the IL-8-induced chemotaxis of CD38KO and WT neutrophils was equivalent (Figure 8D). Thus, these data indicate that CD38KO neutrophils make defective

chemotactic responses to some, but not all, chemoattractants.

[0138] CD38 is expressed and enzymatically active on neutrophils. Since CD38KO neutrophils appear to have an intrinsic defect in chemotaxis, CD38 expression and enzyme activity on mouse and human neutrophils was determined. Neutrophils isolated from the bone marrow and blood of WT mice clearly expressed CD38 (Figure 9 A), and likewise, human peripheral blood neutrophils also expressed CD38 (Figure 9B). Interestingly, when WT mice were injected intraperitoneally with the inflammatory agent, thioglycollate, CD38 expression increased significantly on the neutrophils isolated from the blood and peritoneal cavity (Figure 9D). Next, to test whether CD38-expressing neutrophils can catalyze the cyclase reaction, WT and CD38KO neutrophils were incubated with the NAD+ analogue, nicotinamide guanine dinucleotide (NGD), and then measured the cyclization of NGD into the fluorescent compound cyclic GDP -ribose (Graeff et al., 1994 J. Biol. Chem 269:30260-30267) (cGDPR). As shown in Figure 9C, WT

neutrophils, but not CD38KO neutrophils, produced cGDPR rapidly upon incubation with NGD,

indicating that CD38-expressing neutrophils are competent to produce cyclic nucleotides.

[0139] cADPR and ryanodine induce intracellular calcium release in neutrophils. Since cADPR

induces intracellular calcium release through ryanodine receptor (RyR) gated stores (Galione et

al. 1991 Science 253:1143-1146), it was tested whether the RyR/cADPR calcium signaling

pathway was functional in neutrophils. RT-PCR analysis showed that neutrophils express mRNA

for RyR3 (Sorrentino, V. et al., 1993 TIPS 14:98-103; Hakamata Y. et al, 1992 FEBS Lett

312:229-235), although at levels much lower than seen in the brain (Figure 9D). To test whether

the RyRs expressed by neutrophils were functional, intracellular calcium levels ([Ca2 ]i) were

measured in neutrophils that were permeabilized in calcium-free buffer and then stimulated with

ryanodine (Figure 9E). A small, but reproducible, increase in [Ca2⁺] in ryanodine-stimulated

neutrophils that could be blocked by the RyR inhibitor, ruthenium red was observed (Galione et

al. 1991 Science 253:1143-1146). Next, to test whether cADPR could induce intracellular

calcium release in neutrophils, neutrophils were permalized in calcium-free buffer and then

stimulated the cells with purified cADPR (Figure 9F). A small, but easily detectable, rise in

intracellular free calcium was observed. No calcium release was observed when the cADPR was

first hydrolyzed by heat inactivation (Lee et al., 1989 J. Biol. Chem. 264:1608-1615) or when the

cells were pre-treated with 8-Br-cADPR, an inactive analogue of cADPR that competitively

antagonizes cADPR binding to RyRs (Guse et al., 1994 Annu. Rev. Immunol 12:593-633). The specificity of the antagonist, 8-Br-cADPR, for cADPR mediated calcium release was further

demonstrated by showing that 8-Br-cADPR was unable to block the accumulation of intracellular

free calcium mediated by thapsigargin (Figure 9G). Together, the data demonstrate that

intracellular calcium can be released through RyR and cADPR-mediated mechanism in

neutrophils.

[0140] CD38 catalyzed cADPR is required for extracellular calcium influx in fMLP-activated

neutrophils. Signaling through chemokine/chemoatfractant G-protein coupled receptors such as

FPR and the IL-8 receptors results in increased [Ca2 ]i due to a combination of intracellular calcium release and extracellular calcium influx (Murphy, P.M., 1994 Annu. Rev. Immunol

12:593-633; Demaurex N. et al., 1994 Biochem J. 297:595-601; Schorr W. et al., 1999 Eur. J.

Immunol 29:897-904: Lew et al., 1989 Eur. J. Clin. Invest. 19:338-346). Since CD38KO

neutrophils were defective in chemotaxis assays to fMLP and lacked the ability to produce the

calcium mobilizing metabolite, cADPR, it was hypothesized that calcium mobilization in

response to fMLP would be deficient in CD38KO neutrophils. To test this, CD38KO or WT

neutrophils were stimulated with fMLP or IL-8 in calcium-free media and intracellular calcium

release was measured (Figure 10A). An immediate sharp rise in intracellular calcium was

observed that gradually declined over next 5 minutes in fMLP-stimulated WT neutrophils. hi

contrast, in fMLP-stimulated CD38KO cells, the magnitude of [Ca2⁺]i after fMLP stimulation

was reduced by approximately 20% and the [Ca2⁺]i declined to baseline at least 2 minutes

earlier. Unlike the reduced [Ca2⁺]i found in fMLP-stimulated CD38KO neutrophils, the [Ca2⁺]i of IL-8 stimulated CD38KO and WT neutrophils was identical. Thus, these data suggested that CD38 may be necessary for optimal intracellular calcium release after fMLP, but not IL-8, stimulation.

[0141] Next, to assess whether CD38 was required for extracellular calcium influx, stimulated CD38KO or WT neutrophils were stimulated with fMLP or IL-8 in calcium-containing media (Figure 10B). When we added fMLP to WT neutrophils, a rapid increase in [Ca2⁺]i, due to intracellular calcium release was observed, as well as a second extended, increase in [Ca2⁺]i, due to extracellular calcium influx. In striking contrast, the calcium influx phase of the response was essentially ablated in the fMLP-stimulated CD38KO neutrophils. Interestingly, when WT and CD38KO neutrophils were stimulated with IL-8 in calcium containing media, it was found that IL-8 induced a equivalent immediate increase in [Ca2⁺]i that rapidly declined to baseline levels in both WT and CD38KO neutrophils, indicating that IL-8 did not induce extracellular calcium influx in either WT or CD38KO neutrophils.

[0142] To determine whether cADPR regulates calcium mobilization in fMLP stimulated neutrophils, CD38KO and WT neutrophils were preincubated with increasing concentrations of the cADPR antagonist, 8-Br-cADPR, and then stimulated with fMLP or IL-8 (Figure IOC).

When 8-Br-cADPR-treated WT cells were stimulated with fMLP, the release of intracellular calcium as well as the influx of extracellular calcium was reduced in a dose-dependent fashion to the levels seen in CD38KO cells. In contrast, addition of 8-Br-cADPR to IL-8 stimulated neutrophils had absolutely no effect on the [Ca2⁺]i of either WT or CD38KO neutrophils. Together, these data indicate that CD38 -produced cADPR regulates intracellular calcium release and extracellular calcium influx in response to fMLP, and that neither CD38 nor cADPR are necessary for calcium mobilization in IL-8 stimulated neutrophils.

[0143] CD38 catalyzed cADPR is required for neufrophil chemotaxis to fMLP but not IL-8. To test whether cADPR-mediated calcium mobilization is required for chemotaxis to fMLP, WT neutrophils were preincubated with either EGTA or 8-Br-cADPR and then chemotaxis to fMLP or IL-8 in a checkerboard chemotaxis assay was measured (Figure 10D). When WT neutrophils (no pre-treatment) were incubated with media in the top chamber and fMLP or IL-8 in the bottom chamber, the cells efficiently migrated to the bottom chamber. However, if the extracellular calcium was chelated with EGTA or if the cells were pre-treated with the cADPR antagonist, 8- Br-cADPR, chemotaxis of the WT neutrophils to fMLP was reduced by more than 80%. Importantly, EGTA or 8-Br-cADPR treatment had absolutely no effect on the ability of neutrophils to chemotax to IL-8. Thus, extracellular calcium influx, regulated by cADPR- mediated intracellular calcium release, is necessary for fMLP-induced chemotaxis of neutrophils.

[0144] An analogue of NAD+ inhibits neufrophil chemotaxis to fMLP, but not IL-8, in a CD38- dependent fashion. Since CD38 catalyzed cADPR appeared necessary for neufrophil chemotaxis to fMLP, it was predicted that chemotaxis could be inhibited by treating neutrophils with NAD⁺

analogues that could be converted by CD38 into antagonists of the cADPR signaling pathway.

To test this prediction, neufrophils with pretreated with nicotinamide 8-bromoadenine dinucleotide (N(8-Br-A)D⁺), a substrate that can be converted by CD38 into 8-Br-cADPR, the cADPR antagonist that was used in our earlier experiments. To first test whether N(8-Br-A)D altered extracellular calcium influx in fMLP-activated neutrophils, WT neutrophils were preheated with N(8-Br-A)D⁺, the cells were stimulated with fMLP and then [Ca2⁺]i was measured (Figure 11 A). N(8-Br-A)D pre-treatment inhibited the entry of extracellular calcium in fMLP -treated neutrophils. Next, WT and CD38KO neutrophils were preheated with N(8-Br- A)D⁺ or left in media alone, followed by testing for their ability to chemotax to fMLP (Figure 1 IB) or IL-8 (Figure 1 IC). Untreated WT neutrophils chemotaxed to both fMLP and IL-8, while untreated CD38KO neutrophils could not chemotax to fMLP, but could chemotax to IL-8. Interestingly, pre-treatment of WT neutrophils with N(8-Br-A)D⁺ severely reduced neufrophil chemotaxis to fMLP but had no effect on their ability to chemotax to IL-8. Pre-treatment of the CD38KO neutrophils with N(8-Br-A)D⁺ had no effect on the chemotaxis of the CD38KO neutrophils to either fMLP or IL-8, indicating that the N(8-Br-A)D⁺ induced inhibition of fMLP- mediated chemotaxis was CD38 dependent. Together, the data demonstrate that NAD analogues can regulate calcium responses and chemotaxis of neutrophils in a CD38-dependent fashion.

7. EXAMPLE: MOUSE MODEL OF ALLERGIC LUNG

DISEASE AND ROLE OF CD38

[0145] The subsection below describes data demonstrating that CD38-deficient eosinophils are unable to be recruited to the site of airway inflammation induced by allergens. 7.1. MATERIALS AND METHODS

[0146] OVA priming and sensitization. C57BL/6 WT mice were immunized intraperitoneally

with 20 μg chicken ovalbutnin (OVA) adsorbed to alum. Immunized mice were sacrificed 30

days post-immunization and the OVA-primed CD4 T cells were purified from the spleen using

MACS magnetic beads that were directly conjugated with anti-CD4. Naive CD4 T cells were

purified using anti-CD4 conjugated MACS beads from unimmunized C57BL/6 WT mice. Naive

or OVA-primed T cells were injected intravenously into either C57BL/6 WT or CD38KO

recipients at 1 x 10⁷ CD4 T cells per mouse to generate 4 groups of 10 mice each indicated in

Figure 11 A and B. Recipient mice were then sensitized intratracheally with 10 μg OVA in PBS

on each of 7 consecutive days immediately following T cell transfer. Mice were sacrificed on the

eighth day after T cell transfer, and infiltrating cells were removed from the airways and alveoli

of the lungs by broncheoalveolar lavage as described in Section 6.2.2, supra. Total cells were

then enumerated by counting on a hemocytometer and differential cell counts were performed by

centrifuging cells on to a glass slide, staining with Diff-Quick and identifying at least 200 cells

per slide at 400X.

7.2. RESULTS

[0147] To determine if CD38 controls the recruitment of cells other than neufrophils to the lung,

a mouse model of allergic lung disease that mimics many of the properties of human asthma was

used (Lloyd CM et al., 2001, Adv. Immunol. 77:263-295). An important component of asthma is airway inflammation, which is thought to be induced or exacerbated by the activities of

eosinophils that have been recruited to the lung. Although eosinophils are primarily responsible

for the pathology of asthma (Broide, DH et al, 1991, J. Allergy Clin. Immunol. 88:637-648),

their recruitment and function appears to be controlled by T cells that have been primed to

allergenic antigens (Gavett et al, 1994, Respir. Cell Mol. Biol. 10:587-593). Such T cells often

produce type 2 cytokines, such as IL-4, IL-5 and EL- 13, as well as chemokines like eotaxin

(Cohn, L. et al., 1988, J. Immunol. 161:3813-3816; Drazen JM et al., 1996, J. Exper Med. 183:1-

5). To examine the ability of CD38 to regulate eosinophil recruitment independently of any

effects of CD38 on T cell activation, WT mice were immunized with the antigen OVA. After 30

days, CD4 T cells from these OVA-primed mice were transfened to either WT or CD38KO

recipients. As a control, naive CD4 T cells were transferred from unimmunized WT mice to

either WT or CD38KO recipients. Recipient mice were then sensitized intratracheally with 10

μg of OVA in PBS on each of eight consecutive days immediately following T cell transfer.

Mice were sacrificed on the ninth day after T cell transfer and the cells in the airways of the lungs

were enumerated.

[0148] As seen in Figure 12A, substantial numbers of neutrophils were recruited to the airways

of WT mice regardless of whether they received naive or primed CD4 T cells. In contrast,

although CD38KO trice that received primed T cells did have significantly more neutrophils in

the airways than CD38KO mice that received naive T cells, relatively few neutrophils were

recruited to the airways of CD38KO mice compared to the airways of WT mice. Thus, neufrophil recruitment to the lung in a model of allergic airway disease is also dependent on the

expression of CD38.

[0149] Strikingly, the recruitment of eosinophils to the airways of OVA-sensitized mice was

dependent on the presence of both primed CD4 T cells and the expression of CD38. As seen in

Figure 12B there was a 30-fold reduction in the numbers of eosinophils recruited to the lungs of

CD38K0 mice that had received primed CD4 T cells relative to that in WT mice that had

received primed CD4 T cells and a 10-fold reduction in the numbers of eosinophils recruited to

the airways of CD38KO mice that received naive CD4 T cells relative to that in WT mice that

had received naive CD4 T cells. Therefore, the recruitment of eosinophils to the lung in a model of allergic airway disease is also dependent on CD38.

8. EXAMPLE: CLONING OF SCHISTOSOMA MANSONI

CD38 HOMOLOGUE

[0150] The subsection below describes the cloning and sequencing of a S. mansoni CD38

homologue referred to as SM38. Helminths, such as S. mansoni, are broadly defined as worm

parasites that infect and can cause pathogenesis in most invertebrates, vertebrates and plant

species. The genus Schistosoma consists of parasitic flatworms whose definitive habitat is the

bloodstream of warm-blooded vertebrates. Four species of Schistosoma, including S. mansoni

cause disease in 200-400 million humans per year and kill up to 1 million people each year

(WHO, 1996). Additionally, at least two Schistosoma species infect domesticated cattle and

sheep causing serious economic losses. Thus, it would be beneficial to develop effective antibiotic drugs that could be used to treat infected humans and/or animals. The pathogenesis of

Schistosoma infection is caused mainly by the deposition of eggs by the mature worm into

various tissues and organs of humans and animals where granulomas then form leading to

fibrosis and tissue damage. However, the cercariae (immature worm) and fully mature worm

also release a number of proteins and lipid mediators that can also induce an immune

inflammatory response (Fusco, AC et al., 1991, J. Paristol. 77:649-657). The treatment of choice

in schistosomaisis is the drug praziquantel which appears to induce calcium influx across the

tegument of the worm causing immediate muscle contraction and paralysis (Kohn, AB et al.,

2001, J. Biol. Chem 276:36873-36876). Thus, drugs that modulate schistosome calcium

responses, particularly within the muscle, might be effective in the treatment of this disease.

8.1. MATERIALS AND METHODS

8.1.1. CLONING OF SHISTOSOMA MANSONI CD38 HOMOLOGUE

[0151] Primers were made corresponding to the EST sequence found in Genbank accession

#AW017229. (5' primer: acatctttgtggtactgaatggctcgg and 3' primer: tgagtaatgtctcgacgtttgacctcg). S. mansoni cDNA libraries were obtained from Dr. Phillip

Lo Verde (SUNY, Buffalo), and were subjected to PCR using the primers indicated above. The

library (1-20 μl) and dH2O were heated to 70°C for 10 minutes and were then combined with the

remainder of the PCR reagents and cycled. The cycles were: 95°C 5 minutes, 1 cycle, followed

by 95°C 1 minute, 65°C 1 minute and 72°C 2 minutes for 35 cycles followed by 1 cycle at 72°C for 5 minutes. The expected 330 bp band corresponding to EST AW017229 was isolated, TOPO cloned, and then used as a probe to screen 250,000 plaques from the S. mansoni cDNA library.

Five positives were isolated and then subjected to 3 more rounds of screening in order to produce

plaque pure clones. All five clones were fully sequenced on both strands. The nucleotide

sequence and amino acid translation of four of the clones were identical (referred to as SM38).

The stop codon and polyadenylation sites were identified in all of the SM38 clones, but the

initiation methionine was not present in any of the clones. To obtain the 5' end of the SM38

gene, a single primer extension approach (NAR, 1994, vol 22, No. 16, p3427-3428) was utilized.

A first round of PCR was performed using an external SM38 primer (5'

catcgaataaccctgatttcataacac) and the universal reverse primer for Bluescript. Two μl of this

reaction was then subjected to PCR using an internal nested SM38 primer (5'

gataaagtaagaactcgtgcc) and the universal reverse primer. A 200 and a 300 bp band were

identified from this reaction and were directly sequenced. The sequence obtained overlapped

124 bp with the 5' end of the SM38 clones and included an additional 153 bp of sequence,

however the no stop codon was detected, indicating that we still did not have the 5' end of the

gene. Therefore, classic 5"RACE (PNAS vol 85 pp 8998-9002, Dec. 1998) was performed using

using cDNA prepared from RNA isolated from adult S. mansoni worms (RNA provided by Dr.

P. LoVerde, SUNY Buffalo). 10X Taq buffer, dNTP's, cDNA and Expand High Fidelity Taq

were combined with the dT-AP primer (see ref. For details) and cycled for 5 minutes at 95 C '

followed by 2 minutes at 50 C and 40 minutes at 72 C. After this 40 minute incubation the 5'

external SM38 primer (see above) and AP primers were added and cycled for 35 cycles under the conditions: 95 C for 15 sec, 47 C for 30 sec, 72 C for 2 minutes followed by a 5 minute

extension at 72°C. The reactions were run on a 1.5% agarose gel and a 300 bp band was isolated

using Qiagen Gel -Kit. The 5' RACE product was directly sequenced with the AP and 5' external

SM38 primer. Two potential initiation methionines were identified in the sequence and two stop

codons were found 13-19 amino acids upstream of the methionine residues. The RACE product

was subsequently cloned. All three clones containing SM38 sequence (Two PCR generated

clones and one clone from the S. mansoni cDNA library) were contiguous and overlapping.

When assembled, the SM38 sequence included 1049 bp of sequence including 5' untranslated

sequence, two potential initiation methionines, an open reading frame encoding a 303 amino acid

protein, a stop codon, 3 ' untranslated sequence and a poly-adenylation site.

8.2. RESULTS [0152] Since drugs that modulate calcium responses in the muscle fibers of Schistosomes appear

to be effective anti-helminth reagents (Kohn et al, 2001, J. Biol. Chem. 276:36873-36876), we

set out to identify specific calcium modulating targets of Schistosomes. It has been recently

shown that Schistosomes express Ryanodine Receptors (RyR) within their muscle fibers (Day et

al., 2000, Parasitol. 120:417-420; Silva et al., 1998, Biochem. Pharmacol. 56:997-1003).

Agonists of RyRs expressed in vertebrate smooth and skeletal muscle are known to regulate

intracellular calcium release, voltage gated calcium influx and muscle contractility. Interestingly,

S. mansoni muscle fibers treated with RyR agonists such as caffeine induced the release of intracellular calcium and induced contraction of the muscle fiber. Although drugs such as

caffeine can modulate RyR-dependent calcium responses, the physiological modulator of RyRs,

at least in vertebrate muscle fibers is cyclic ADP-ribose (cADPR). cADPR is a known calcium

mobilizing metabolite that is produced by ADP-ribosyl cyclase enzymes such as the mammalian

CD38 protein and the invertebrate Aplysia cyclase enzyme. To determine whether Schistosomes

express an enzyme capable of producing the calcium mobilizing second messenger, cADPR, a

search was performed of the publicly available EST sequences looking for Schistosome

sequences that when translated would have homology to the mammalian CD38 and Aplysia

enzymes. Three EST sequences (EST AI067047, EST AW017229 and EST N20756) were

identified that could be assembled into a contiguous and overlapping sequence (Fig 13). This

assembled sequence shared limited but significant homology with both CD38 and the Aplysia cyclase enzymes.

[0153] To determine whether the assembled ESTs actually represented an authentic cDNA,

primers were prepared from the sequence of EST A WO 17229 and performed PCR on a S.

mansoni cDNA library. A 330 base pair fragment was isolated from the PCR reaction and was

sequenced. As expected the sequence of the fragment matched that of the EST. The fragment

was then used as a probe to screen 250,000 plaques from the S. mansoni cDNA library. Five

independent plaques which hybridized to the EST probe were isolated, plaque purified and

sequenced on both DNA strands. The sequence information was then used to design additional

primers to isolate the 5' end of the cDNA (see methods). The complete cDNA sequence isolated from the S. mansoni library was then assemble and compared to the ESTs. The alignment,

shown in Figure 13, indicates that the contiguous assembly of the EST sequences was correct and

that the cloned cDNA (refereed to as SM38) included at least an additional 421 base pairs of

sequence not found in any EST. Translation of the DNA sequence gave rise to a 299 amino acid

sequence (Figure 14) containing structural motifs typical of cyclase enzymes (Prasad, GS, 1996

Nature Struct, biol. 3:957-964). In particular, the SM38 protein contains conserved amino acid

residues that align with critical catalytic and active site residues found in the Aplysia cyclase

enzyme (Munshi C, et al., 1999, J. Biol. Chem. 274:30770-30777) and in mammalian CD38

(Munshi C, et al., 2000, J. Biol. Chem. 275:21566-21571; Graeff R., 2001, J. Biol. Chem.

276:12169-12173)(Figure 15 A-B). Additionally, cysteine residues that are critical for the

assembly of the tertiary structure of the cyclase enzymes (Prasad GS, et al., 1996, NAture Struct,

biol. 3:957-964)are also conserved in SM38 ; (Figure 15A-B). Importantly, the SM38 cDNA

sequence encodes for a complete cyclase enzyme domain (Figure 16).

[0154] Based on these results, we have shown that Schistosomes such as S. mansoni encode a

protein (SM38) that is highly homologous at the structural level to enzymes that are capable of

catalyzing the production of the calcium mobilizing second messenger, cADPR. Since

Schistosomes also express RyRs which release intracellular calcium in response to cADPR, it is

predicted that SM38 will be able to regulate calcium response in Schistosomes. Furthermore,

since regulation of calcium influx, particularly in Schistosome muscle fibers can result in

paralysis and clearance of the worm, we predict that agonists/antagonists of the SM38 and RyR pathways in Schistosomes may be effective as anti-helminth drugs.

[0155] The present invention is not to be limited in scope by the specific embodiments described

herein which are intended as single illustrations of individual aspects of the invention, and

functionally equivalent methods and components are within the scope of the invention. Indeed,

various modifications of the invention, in addition to those shown and described herein will

become apparent to those skilled in the art from the foregoing description and accompanying

drawings. Such modifications are intended to fall within the scope of the claims. Various

publications are cited herein, the contents of which are hereby incorporated, by reference, in their entireties.

Claims (34)

We Claim:

1. An isolated nucleic acid molecule comprising a nucleotide sequence that

encodes the amino acid sequence shown in Figure 14.

2. The isolated nucleic acid molecule of claim 1 comprising the DNA

sequence of Figure 14.

An isolated nucleic acid molecule comprising the DNA sequence of Figure

14.

4. The isolated nucleic acid molecule of claim 3 comprising a nucleotide

sequence that encodes the amino acid sequence shown in Figure 14.

5. An isolated nucleic acid molecule comprising a nucleotide sequence that

hybridizes to the nucleotide sequence of Claim 1 or 3 under stringent conditions and encodes a

functionally equivalent gene product.

6. An isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes to the nucleic acid of claim 1 or 3 under moderately stringent conditions and encodes

a functionally equivalent SM38 gene product.

7. An isolated nucleic acid molecule that is a SM38 antisense molecule.

8. An isolated polypeptide comprising the amino acid sequence of Figure 14.

9. An isolated polypeptide comprising the amino acid sequence of Figure 14.

10. An isolated polypeptide comprising the amino acid sequence encoded by a

nucleotide sequence that hybridizes to the nucleotide sequence of Claim 1 or 3 under stringent

conditions and encodes a functionally equivalent gene product.

11. An isolated polypeptide comprising the amino acid sequence encoded by a

nucleotide sequence that hybridizes to the nucleotide sequence of Claim 1 or 3 under moderately

stringent conditions and encodes a functionally equivalent gene product.

12. A purified fragment of a SM38 protein comprising the cyclase domain of

the SM38 protein.

13. A chimeric protein comprising a fragment of a SM38 protein consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second protein, in

which the second protein is not a SM38 protein.

14. An antibody which is capable of binding a SM38 protein.

15. A recombinant cell containing the nucleic acid of claim 5 or 6.

16. A method of producing a CD38 protein comprising growing a recombinant

cell containing the nucleic acid of claim 5 or 6 such that the encoded CD38 protein is expressed

by the cell, and recovering the expressed CD38 protein.

17. A method for identifying a compound that activates CD38 enzyme activity

comprising (i) contacting a cell expressing CD38 with a test compound in the presence of

substrate and measuring the level of CD38 activity; (ii) in a separate experiment, contacting a cell

expressing CD38 protein with a vehicle control in the presence of substrate and measuring the

level of CD38 activity where the conditions are essentially the same as in part (i), and then (iii)

comparing the level of CD38 activity measured in part (i) with the level of CD38 activity in part

(ii), wherein an increased level of CD38 activity in the presence of the test compound indicates

that the test compound is a CD38 activator.

18. A method for identifying a compound that inhibits CD38 enzyme activity

comprising (i) contacting a cell expressing CD38 with a test compound in the presence of a chemoattractant and substrate and measuring the level of CD38 activity; (ii) in a separate

experiment, contacting a cell expressing CD38 and subsfrate and measuring the level of CD38

activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the

level of CD38 activity measured in part (i) with the level of CD38 activity in part (ii), wherein a

decrease level of CD38 activity in the presence of the test compound indicates that the test

compound is a CD38 inhibitor.

19. The method of claim 17 or 18 further comprising the presence of a

chemoattractant in step (i) and (ii) and wherein the cell expressing CD38 expresses a

chemoattractant receptor.

20. The method of claim 17 or 18 wherein CD38 ADP-ribosyl cyclase activity

is measured.

21. The method of claim 17 or 18 wherein levels of c ADPR are measured.

22. The method of claim 17 or 18 wherein the level of NAADP is meausred.

23. The method of claim 17 or 18 wherein intracellular calcium levels are measured.

24. The method of claim 17 or 18 wherein CD38 mediated cell migration is

measured.

25. A method for identifying a compound that modulates the activity of CD38

comprising the steps of:

(i) contacting a test compound with a CD38 protein;

(ii) detennining whether said compound binds to the CD38 protein;

(iii) and selecting a test compound that binds to said CD38 protein as being a

compound that can be used to modulate the activity of the CD38 protein.

26. A method for identifying a compound that activates SM38 enzyme activity

comprising (i) contacting a cell expressing SM38 with a test compound in the presence of

substrate and measuring the level of SM38 activity; (ii) in a separate experiment, contacting a

cell expressing SM38 protein with a vehicle control in the presence of substrate and measuring

the level of SM38 activity where the conditions are essentially the same as in part (i), and then

(iii) comparing the level of SM38 activity measured in part (i) with the level of SM38 activity in

part (ii), wherein an increased level of SM38 activity in the presence of the test compound

indicates that the test compound is a SM38 activator.

27. A method for identifying a compound that inhibits SM38 enzyme activity comprising (i) contacting a cell expressing SM38 with a test compound in the presence of a

chemoattractant and substrate and measuring the level of SM38 activity; (ii) in a separate

experiment, contacting a cell expressing SM38 and substrate and measuring the level of SM38

activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the

level of SM38 activity measured in part (i) with the level of SM38 activity in part (ii), wherein a decrease level of SM38 activity in the presence of the test compound indicates that the test

compound is a SM38 inhibitor.

28. The method of claim 26 or 27 wherein levels of cADPR are measured.

29. The method of claim 26 or 27 wherein the level of NAADP is meausred.

30. The method of claim 26 or 27 wherein intracellular calcium levels are

measured.

31. The method of claim 26 or 27 wherein CD38 mediated cell migration is measured.

32. A method for identifying a compound that modulates the activity of SM38 comprising the steps of:

(i) contacting a test compound with a SM38 protein; (ii) determining whether said compound binds to the SM38 protein; (iii) and selecting a test compound that binds to said SM38 protein as being a compound that can be used to modulate the activity of the SM38 protein.

33. A method of modulating the migratory activity of cells expressing CD38 comprising contacting said cells with a CD38 inhibitor.

34. A method of modulating the migratory activity of cells expressing CD38 comprising contacting said cells with a CD38 activator.