1. PHL 616 Drug Discovery & Development
Fourth Lecture By Abdelkader Ashour, Ph.D. Phone:

2. Creation of a new drug, Case histories
Omeprazole (Losec)
In 1966, Astra started a project aimed at developing inhibitors of gastric acid secretion, having previously developed profitable antacid preparations
They started a chemistry program, and collaborated with an academic group to develop a suitable in vivo screening assay in rats
Compounds with weak activity were quickly identified; initial hepatotoxicity problems were overcome, and a potential development compound was tested in humans in 1968.
It had no effect on acid secretion, and the project narrowly escaped termination
At the same time, Searle reported a new class of inhibitory compounds, benzimidazoles, which were active but toxic
Astra began a new chemistry program based on this series, and in1973 produced a highly active compound which was proposed for further development

3. Creation of a new drug, Case histories
Omeprazole, contd.
Unfortunately, Astra found that a Hungarian company had a patent on this compound. However, Astra found that the Hungarian patent had actually expired because the company had defaulted on payment of the fees to the patent office!
Further studies with this compound revealed problems with thyroid toxicity, however, and more demands to terminate this hapless project were narrowly fought off
Thyroid toxicity was thought to be associated with the thiouracil structure, and further chemistry aimed at eliminating it resulted in the synthesis of picoprazole, the forerunner of omeprazole, in 1976
After yet another toxicological alarm –this time vasculitis in dogs– which turned out to be an artifact, picoprazole was tested in human patients suffering from Zollinger–Ellison syndrome and was found to be highly effective in reducing acid secretion

4. Creation of a new drug, Case histories
Omeprazole, contd.
At around the same time, an academic group showed that acid secretion involved a specific transport mechanism, the proton pump, which was strongly inhibited by picoprazole, so its novel mechanism of action was established
Omeprazole, an analogue of picoprazole, was synthesized in 1979, and was chosen for development instead of picoprazole
The chemical development of omeprazole was complicated by its poor stability and sensitivity to light, requiring special precautions in formulation
Phase II/III clinical trials began in 1981, but were halted for 2 years as a result of yet another toxicology scare – carcinogenicity – which again proved to be a false alarm. Omeprazole was finally registered in 1988

5. Creation of a new drug, Case histories
Imatinib (Gleevec)
Imatinib exemplifies the shift towards defined molecular targets that has so altered the approach to drug discovery over the last 20 years
In the mid-1980s, it was discovered that chronic myeloid leukaemia (CML), was almost invariably associated with the expression of a specific oncogene product, Bcr-Abl kinase
The enhanced tyrosine kinase activity of this mutated protein was shown to underlie the malignant transformation of the white blood cells
The proven association between the gene mutation, the enhanced kinase activity and the distinct clinical phenotype, provided a particularly clear example of cancer pathogenesis

6. Creation of a new drug, Case histories
Imatinib (Gleevec)
Tyrosine kinases are enzymes that transfer phosphate from ATP to tyrosine residues on substrate proteins that in turn regulate cellular processes such as proliferation, differentiation, and survival
Therefore it is not surprising that deregulated tyrosine kinase activity has a central role in malignant transformation. This has also made tyrosine kinases attractive therapeutic targets for pharmacologic inhibition
On this basis, the oncology team of Ciba-Geigy (later Novartis) began a project seeking specific inhibitors of Abl kinase
Imatinib (signal transduction inhibitor 571; STI571, formerly CGP57148B) is a rationally designed abl-specific tyrosine kinase inhibitor

7. Imatinib, Philadelphia chromosome and bcr-abl
A gene formed when pieces of chromosomes 9 and 22 break off and trade places. The abl proto-oncogene from chromosome 9 joins to sequences from chromosome 22, the breakpoint cluster region (bcr) on chromosome 22, to form the bcr-abl fusion gene
The changed chromosome 22 with the fusion gene on it is called the Philadelphia chromosome. The bcr-abl fusion gene is found in most patients with chronic myelogenous leukemia (CML), and in some patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML)

8. Imatinib, Philadelphia chromosome and bcr-abl
c-abl, the cellular homologue of the transforming protein found in the Abelson murine leukemia virus (v-abl), encodes for a non-receptor tyrosine kinase
The c-abl protein has tightly regulated kinase activity and shuttles between the nucleus and cytoplasm, whereas the bcr-abl fusion proteins are exclusively cytoplasmic and have enhanced tyrosine kinase activity that is essential for their transforming ability

9. Imatinib, Bcr-Abl Activity

10. Imatinib, Bcr-Abl Activity

11. Common intracellular signaling proteins
GTP-binding proteins with GTPase activity function as molecular switches.
When bound to GTP they are active; when bound to GDP, they are inactive.
They fall into two categories, trimeric G proteins and Ras-like proteins.
Protein kinases: modulate the activity or the binding properties of substrate proteins by phosphorylating serine, threonine, or tyrosine residues.
The phosphorylated form of some proteins is active, whereas the dephosphorylated form of other proteins is active.
The combined action of kinases and phosphatases can cycle proteins between active and inactive states.
Adapter proteins contain various protein-binding motifs that promote the formation of multiprotein signaling complexes.

12. 3. Kinase-linked Receptors, General structure & activation of receptor tyrosine kinases
Tyrosine-kinase (called receptor tyrosine kinase, more common) and guanylate cyclase-linked (much less common) receptors
Actions: take minutes
Examples: Growth factors, hormones (e.g. insulin) and cytokines
Receptors for various hormones (e.g., insulin) and growth factors possess tyrosine kinase activity in their intracellular domain.
The intracellular domain incorporates both ATP- and substrate binding sites
Cytokine receptors do not usually have intrinsic kinase activity, but associate, when activated by ligand binding, with kinases known as Jaks, which is the first step in the kinase cascade

13. 3. Kinase-linked Receptors, General structure and activation of receptor tyrosine kinases
The ligands for some RTKs, such as the receptor for EGF, are monomeric; ligand binding induces a conformational change in receptor monomers that promotes their dimerization.
The ligands for other RTKs are dimeric; their binding brings two receptor monomers together directly.
In either case, upon ligand binding, a tyrosine kinase activity is “switched on” at the intracellular portion.
The kinase activity of each subunit of the dimeric receptor initially phosphorylates tyrosine residues near the catalytic site in the other subunit.
Subsequently, tyrosine residues in other parts of the cytosolic domain are autophosphorylated.
Protein phosphorylation leads to altered cell function via the assembly of other signal proteins

14. 3. Kinase-linked Receptors, Activation of Ras following binding of a hormone (e.g., EGF) to an RTK
The adapter protein GRB2 binds to a specific phosphotyrosine on the activated RTK and to Sos, which in turn interacts with the inactive Ras·GDP
The guanine nucleotide – exchange factor (GEF) activity of Sos then promotes formation of active Ras·GTP
Note that Ras is tethered to the membrane by a farnesyl anchor

15. 3. Kinase-linked Receptors, Kinase cascade that transmits signals downstream from activated Ras protein
Activated Ras binds to the N-terminal domain of Raf, a serine/threonine kinase
Raf binds to and phosphorylates MEK, a dual-specificity protein kinase that phosphorylates both tyrosine and serine residues
MEK phosphorylates and activates MAP kinase, another serine/threonine kinase
MAP kinase phosphorylates many different proteins, including nuclear transcription factors, that mediate cellular responses

16. 3. Kinase-linked Receptors, Growth Factor Receptors
Conformation change
Dimerisation
Tyrosine autophosphrylation
Phosphorylation of Grb2
Activation of Ras GDP/GTP Exchange
MEMBRANE
Ras
Grb2
GTP +
Activation
Sos
Tyrosine residue
Raf
Grb2
Mek
Binding of SH2-domain protein (Grb2)
Agonist binding leads to dimerisation and autophosphorylation of the intracellular domain of each receptor
SH2 domain proteins, Grb2, then bind to the phosphorylated receptor and are themselves phosphorylated
Ras, which is a proto-oncogene product, functions like a G-protein, and conveys the signal (by GDP/GTP exchange)
Activation of Ras in turn activates Raf, which is the first of a sequence of three kinases, each of which phosphorylates, and activates, the next in line
The last of these, mitogen-activated protein (MAP) kinase, phosphorylates one or more transcription factors that initiate gene expression, resulting in a variety of cellular responses, including cell division
MAP kinase
Various transcription
factors
NUCLEUS
Gene Transcription

17. 3. Kinase-linked Receptors, CytokinesCytokine ReceptorsB
Cytokine
Conformation change
& Binding of Jak
Phosphorylation of receptor + Jak
MEMBRANE
Jak
Jak
Jak
Jak
Cytokine binding leads to receptor dimerisation, and this attracts a cytosolic tyrosine kinase unit (Jak) to associate with, and phosphorylate, the receptor dimer
Among the targets for phosphorylation by Jak are a family of transcription factors (Stats) which bind to the phosphotyrosine groups on the receptor-Jak complex, and are themselves phosphorylated and dimerized
Thus activated, Stat migrates to the nucleus and activates gene expression
Stat
Binding & phosphorylation of SH2-domain protein (Stat)
Dimerization of Stat
Stat
Stat
NUCLEUS
Gene Transcription

18. Imatinib, Bcr-Abl Activity