1. 성균관대학교 약학과
김연수

2. Contents
Introduction
Results
Discussion and Conclusions

3. Phosphatase
Phosphatase enzymes are essential to many biological functions, because phosphorylation and dephosphorylation
serve diverse roles in cellular regulation and signaling
Kinase vs. Phosphatase
Whereas phosphatases remove phosphate groups from molecules, kinases catalyze the transfer of phosphate groups to molecules from ATP
<Phosphatase action>
<Kinase action>

4. Phosphatase 1. Protein phosphatase (PP)
- enzyme that dephosphorylates an amino acid residue of its protein substrate
- phosphatases remove the phosphate group
Protein phosphatase
Tyrosine phosphatase (PTP)
Serine/Threonine phosphatase
Dual specificity phosphatase
Histidine phosphatase
protein kinases act as signaling molecules by phosphorylating proteins, phosphatases remove the phosphate group, which is essential if the system of intracellular signaling is to be able to reset for future use.
Gluconeogenesis is a biosynthetic pathway wherein glucose is created from noncarbohydrate precursors; the pathway is essential because many tissues can only derive energy from glucose
glucose-6-phosphatase and fructose-1,6-bisphosphatase catalyze irreversibe steps in gluconeogenesis 

5. Phosphatase 2. Nucleotidases 3. In gluconeogenesis (포도당 신생합성)
- enzyme that catalyzes the hydrolysis of a nucleotide, forming a nucleoside and a phosphate ion.
3. In gluconeogenesis (포도당 신생합성)
- Phosphatases can also act on carbohydrates, such as intermediates in gluconeogenesis.
- glucose-6-phosphatase and fructose-1,6-bisphosphatase 
protein kinases act as signaling molecules by phosphorylating proteins, phosphatases remove the phosphate group, which is essential if the system of intracellular signaling is to be able to reset for future use.
Gluconeogenesis is a biosynthetic pathway wherein glucose is created from noncarbohydrate precursors; the pathway is essential because many tissues can only derive energy from glucose
glucose-6-phosphatase and fructose-1,6-bisphosphatase catalyze irreversibe steps in gluconeogenesis 

6. SHP (Src Homology Phosphatase)
SHP2 (SH2 protein tyrosine phosphatase – non-receptor protein tyrosine phosphatase
encoded by the PTPN11 gene
→ SH2 (Src Homology 2 domains) + PTP (protein tyrosine phosphatase)
Mutation in PTPN11 ① Germline mutation - Noonan syndrome(50%), Leukemia…..
② Somatic mutation - Myeloid malignance, Solid tumor….
Cytoplasmic tyrosine phosphatase
Binding to protein – SH2 The function of SH2 domains is to specifically recognize the phosphorylated state of tyrosine residues →the fundamental event of signal transduction
Catalysis of protein tyrosine phosphorylation-PTP
Protein tyrosine phosphatases (PTPs) are essential regulators of signal transduction pathways, and control,

7. SHP - cell signaling
SHP2 has an important role in signal transduction downstream of growth factor receptor signaling and was the first reported oncogenic tyrosine phosphatase
SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signaling pathway
In cells, SHP2 function in the cytoplasm downstream of multiple receptor-tyrosine kinases and is involved in numerous oncogenic cell signaling cascades
has been reported involved in insulin resistance through PI3K/Akt/mOTR signaling
has also been considered to play a vital role in carcinogenesis via Ras/Erk pathways

8. SHP - cell signaling
SHP2 also has been reported to participate in the T cell programed cell death/checkpoint pathway (PDL1/PD-1) and contribute to immune evasion.
Activated PD-1 recruits SHP2 to the membrane where it dephosphorylates costimulatory receptor CD28 and suppresses T-cell function

9. SHP-scaffold
1. Salicylic acid-based Hydroxyindole carboxylic acid-based
- anticancer and antileukemia therapy Targeting the Oncogenic SHP2 inhibitor
3. Cryptotanshinone Oxindole-based
- selectively and potently inhibiting ) Shp2 over Shp1 in vitro
5. Cefsulodin-based
- third generation β-lactam cephalosporin antibiotic
- exhibits SHP2 inhibitory activity
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
J. Med. Chem. 2014, 57, 6594−6609
J. Med. Chem. 2013, 56, 7212−7221
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
ACS Med. Chem. Lett. 2015, 6, 782−786

10. SHP – Binding site Allosteric site - https://youtu.be/ApKM-IkSElY
ACS Chem. Biol. 2018, 13, 647−656

11. SHP2 inhibitor
Compound 1 proved to be a selective and moderately potent SHP2 inhibitor – allosteric site 1
High aqueous solubility, selectivity, and oral bioavailability enabled in vivo
Compound 2 proved to be a weak SHP2 inhibitor (SHP21‑525 IC50 = 60 μM) – allosteric site 2
poor aqueous solubility (0.047 mM in pH 6.8 buffer) and high lipophilicity (cLogP = 3.9)
The carboxylic acid functionality improved the aqueous solubility of 8 (0.895 mM in pH 6.8 buffer) and 9 (0.535 mM in pH 6.8 buffer) as compared to 2 (0.047 mM in pH 6.8 buffer).
ACS Chem. Biol. 2018, 13, 647−656

12. Introduction

13. Results <Method for phosphatase activity>
November 17, 2016, EMBO reports
<Method for phosphatase activity>
Fluorescence-based assay – DIFMUP assay
Phosphopeptide-based assay
Computer docking
Cell-based assay

14. Results

15. Results

16. Results

17. Results - synthesis

18. Results – synthesis mechanism
Step 1. Suzuki coupling reaction
Step 2. Nucleophilic Aromatic substitution
Step 3. Boc deprotection

19. Results

20. Discussion and Conclusions
A high throughput screen was designed and conducted on full length SHP2 in order to identify allosteric inhibitors.
This methodology allowed for the identification of aminopyrimidine 2.
These studies culminated in the identification of pyrazine 1, a potent, selective, highly soluble, orally bioavailable, and efficacious SHP2 inhibitor exhibiting dose-dependent pathway inhibition and antitumor activity in xenograft models.
Significantly, this new molecular tool 1 should enable further interrogation of the multifaceted roles of SHP2 in general signal transduction and related molecular pathologies.