성균관대학교 약학과 김연수 2018.10.11

Contents Introduction Results Discussion and Conclusions

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>

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 

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 

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,

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

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

SHP-scaffold 1. Salicylic acid-based 2. Hydroxyindole carboxylic acid-based - anticancer and antileukemia therapy - Targeting the Oncogenic SHP2 inhibitor 3. Cryptotanshinone 4. 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

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

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

Introduction

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

Results

Results

Results

Results - synthesis

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

Results

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.