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L. DEDICATED MOLECULAR BIOLOGY AND PROTEIN PURIFICATION GROUPS ARE ESSENTIAL

L. DEDICATED MOLECULAR BIOLOGY AND PROTEIN PURIFICATION GROUPS ARE ESSENTIAL

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2

Src Homology-2 Domains

and Structure-Based,

Small-Molecule Library Approaches

to Drug Discovery

Chester A. Metcalf III and Tomi Sawyer

ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A.



The elucidation of cell-receptor-associated signal transduction pathways

by means of the tools of biochemistry and molecular genetics has

resulted in the identification of a multitude of protein targets for

therapeutic intervention (Table 1) [1]. The fact that many of these

targets have x-ray crystallography and/or NMR spectroscopy to guide

the syntheses of structurally biased single analogues and combinatorial

libraries has ushered the pharmaceutical industry into a new era of drug

discovery. Within cells there exists an enormously diverse data set of

functional proteins and signaling pathways, involving both noncatalytic

and catalytic processes, which are orchestrated through highly specific

protein–protein interactions. In principle, such interactions can be

disrupted or promoted, either directly or indirectly (via enzyme inhibition), through small-molecule intervention driven by structure-based

methods. This chapter discusses the role of Src homology-2 (SH2)

domains as mediators of protein–protein interactions in signal transduction, with a focus on the therapeutic implications of blocking the

SH2 domain of the nonreceptor protein tyrosine kinase Src with



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 1 List of Possible Protein Targets for Therapeutic Intervention

G-Protein-Coupled/Integrin

Receptors

Angiotensin (AT1, AT2)

Bradykinin (B1, B2)

Cholecystokinin (CCKA)

Gastrin (CCKB)

Endothelin (ETA, ETB)

a–Melanotropin (MCR1)

Adrenocorticotropin (MCR2)

Substance P (NK1)

Neurokinin-A (NK2)

Neurokinin-B (NK3)

y-opioid (Enkephalin)

A-opioid (Endorphin)

n-opioid (Dynorphin)

Oxytocin

Somatostatin (sst1–sst5)

Vasopressin (V1A, V1B)

Neuropeptide-Y (Y1-Y5)

Calcitonin



Receptor/Nonreceptor

Kinases/Phosphatases

Receptor tyrosine kinases

Epidermal growth factor

Fibroblast growth factor

Insulin

Nerve growth factor

Platelet-derived growth factor

Nonreceptor tyrosine kinases

Src and Src family (Lck, Hck)

Abl, Syk, Zap-70

Receptor serine/threonine kinases

Transforming growth factor

Nonreceptor serine/threonine kinases

cAMP-Dependent protein kinase

Phosphoinositol-3-kinase (P13K)

Cyclin-dependent kinases (CDKs)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Transcription

Factors/Proteases

NF-nB, STAT, NFAT, SMAD, CREB

Proteases

Aspartic proteases

Pepsin

Renin

Cathepsins (D, E)

HIV-1 protease

Serinyl proteases

Trypsin

Thrombin

Chymotrypsin-A

Kallikrein

Elastase

Tissue plasminogen activator

Factor Xa



Adenosine (A1-A3)

Cathecholamine (a1, a2, h1-h3)

Histamine (H1, H2)

Muscarinic acetylcholine

Seratonin (5HT1-5HT7)

Melatonin (ML1A, ML1B)

Dopamine (D1, D2, D4, D5)

g-Amino butyric acid (GABAB)

Leukotrienes (LTB4, LTC4, LTD4)

Cell adhesion integrin receptors

avh3 (Fibrinogen)

aIIbh3 or gpIIaIIIb (Fibrinogen)

a5h1 (Fibrinectin)

a4h1 (VCAM-1)



Mitogen-activated protein kinase

Protein kinase C (PKC)

Janus family kinases (JAKs)

InB family kinases (IKKs)

Receptor/nonreceptor phosphatases

Receptor tyrosine phosphatases

CD45, LAR



Cysteinyl proteases

Cathepsins (B, H, K, M, S, T)

Proline endopeptidase

Interleukin-converting enzyme

Apopain (CPP-32)

Picornavirus C3 protease

Calpains



Metallo proteases

Exopeptidase group

Peptidyl dipeptidase-A (ACE)

Aminopeptidase-M

Nonreceptor serine/threonine phosphatases Carboxypeptidase-A

PP-1

Calcineurin

Endopeptidase group

VH1

Endopeptidases (24, 11, 24, 15)

Stromelysin

Gelatinases (A, B)

Collagenase

Nonreceptor tyrosine phosphatases

PTP1B, Syp



Source: Ref. 1.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



designed, nonpeptide small molecules. We highlight ARIAD’s approach

to drug discovery by means of structure-based methods and parallel

synthetic libraries to develop cell-active, in vivo effective inhibitors of Src

SH2-dependent signal transduction pathways, leading to novel drugs for

the treatment of osteoporosis.



I. SIGNAL TRANSDUCTION AND PROTEIN–PROTEIN

INTERACTIONS

The network of protein–protein interactions that define signal transduction pathways in most cells originates at a cellular receptor and is

triggered by the binding of specific external stimuli (e.g., growth factors,

antigens, hormones). Such signal transduction pathways are then propagated within the cell to the nucleus resulting in specific gene activation

and protein synthesis (Fig. 1) [2]. Listed in Table 2 are the modular

domains [3] of various signal transduction proteins and the potential

disease areas providing opportunity for therapeutic intervention through

small-molecule inhibitory drugs [4]. For Src, these disease areas are

osteoporosis and cancer. There are more than 50 known SH2-containing

proteins, of which Src was the first to be identified [5]. The SH2 domain

of Src consists of approximately 100 amino acids and binds cognate



Figure 1 Representation of signal transduction pathways describing highly

specific protein–protein interactions.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 2 Signal Transduction Proteins as Potential Therapeutic Targets

Protein (Domains)

Src (SH3-SH2-kinase)

Hck (SH3-SH2-kinase)

Syk (SH2-SH2-kinase)

Zap70 (SH2-SH2-kinase)

Syp (SH2-SH2-phosphatase)

STATs (DNA binding-SH3-SH2)

Grb2 (SH3-SH2-SH3)

p85/PI3K [SH3-SH2-SH2 (p85 subunit)]

Bcr/Abl (SH3-SH2-kinase)



Disease area

Osteoporosis, cancer

Immune disease, AIDS

Allergy, asthma

Autoimmune disease

Anemia

Inflammatory disease

Cancer, chronic myelogenous leukemia

Cancer

Chronic myelogenous leukemia



Source: Ref. 4.



phosphotyrosine(pTyr)-containing proteins as well as synthetic peptides in

a sequence-dependent manner [6]. In addition to the SH2 domain, Src

possesses one SH3 domain (f 60 amino acids), which is characterized by

its affinity for proline-rich sequences, and a bilobed tyrosine kinase

catalytic domain (f 300 amino acids) containing N-terminal (NT) and

C-terminal (CT) domains (Fig. 2) that specifically phosphorylates tyrosine

residues of cognate substrate proteins.



II. Src TYROSINE KINASE AND OSTEOPOROSIS

Molecular insight into the protein conformation states of Src kinase has

been revealed in a series of x-ray crystal structures of the Src SH3–SH2–

kinase domain that depict Src in its inactive conformation [7]. This form

maintains a ‘‘closed’’ structure, in which the tyrosine-phosphorylated

(Tyr527) C-terminal tail is bound to the SH2 domain (Fig. 2). The x-ray

data also reveal binding of the SH3 domain to the SH2–kinase linker

[adopts a polyproline type II (PP II) helical conformation], providing

additional intramolecular interactions to stabilize the inactive conformation. Collectively, these interactions cause structural changes within the

catalytic domain of the protein to compromise access of substrates to the

catalytic site and its associated activity. Significantly, these x-ray structures

provided the first direct evidence that the SH2 domain plays a key role in

the self-regulation of Src.

The bone disease osteoporosis results when an imbalance occurs in

the normal course of bone remodeling, a dynamic and highly regulated



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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