Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4-d]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

Figures & data

Table 1. Sequences and accession numbers for primers (forward, FOR; reverse, REV) used in qPCR

Gene	Sequences for primers	Accession no.
Tnfa	FOR: AGGGTCTGGGCCATAGAACT	NM_013693
	REV: CCACCACGCTCTTCTGTCTAC
Il1b	FOR: GGTCAAAGGTTTGGAAGCAG	NM_008361
	REV: TGTGAAATGCCACCTTTTGA
Il6	FOR: CCACGGCCTTCCCTACTTC	NM_031168
	REV: TTGGGAGTGGTATCCTCTGTGA
Icam1	FOR: AACAGTTCACCTGCACGGAC	NM_010493
	REV: GTCACCGTTGTGATCCCTG
Cxcl1	FOR: AATGAGCTGCGCTGTCAGTG	NM_008176
	REV: TGAGGGCAACACCTTCAAGC
Ccl2	FOR: ACCGACAACAGGAAGTGGAG	NM_009140
	REV: TGGACGTTTCACACAGTGGT
Ho1	FOR: AGGTACACATCCAAGCCGAGA	NM_010442
	REV: CATCACCAGCTTAAAGCCTTCT
Nqo1	FOR: AGGATGGGAGGTACTCGAATC	NM_008706
	REV: TGCTAGAGATGACTCGGAAGG
Nrf2	FOR: CTTTAGTCAGCGACAGAAGGAC	NM_010902
	REV: AGGCATCTTGTTTGGGAATGTG
Gapdh	FOR: CGTCCCGTAGACAAAATGGT	NM_008084
	REV: TTGATGGCAACAATCTCCAC

Figure 1. Drug design and identification of SPA7012 as PAK4 inhibitor. (A) Design strategy for novel PAK4 selective inhibitors. (B) Enzymatic IC₅₀ curves for SPA7012, SPA7016 and SPA7017. (C) Representative structure of pyrido[3,4-d]pyrimidine derivatives. (D) Predicted binding mode of SPA7012 (PDB code: 4O0V). Original ligand GNE-2861 (green) and SPA7012 (brown) are presented in the stick model. The hydrogen bonds are indicated as a green dashed line.

Scheme 1. Synthesis of 1-aminopyrido[4,3-b]indole derivatives. Reagents and conditions: (a) trimethyl orthoacetate, 100 °C; (b) DMF-DMA, MeOH, reflux; (c) 48% HBr, AcOH, room temperature; (d) conc. HCl, 160 °C; (e) phenylhydrazine, Ph₂O, 240 °C; (f) POCl₃, reflux; (g) p-methoxybenzylamine, 200 °C; (h) 1-bromo-3-iodobenzene, Cu, K₂CO₃, 18-crown-6, 1,2-dichlorobenzene, 180 °C; (i) TFA, 60 °C; and (j) R-prop-2-yn-1-ol, Pd(PPh₃)₂Cl₂, TEA/DMSO (2:1), 70 °C.

Scheme 2. Synthesis of the 4-aminopyrazolo[3,4-d]pyrimidine derivatives. Reagents and conditions: (a) trimethyl orthoacetate, 100 °C; (b) hydrazine monohydrate, EtOH, 80 °C; (c) formamide, 180 °C; (d) 1-bromo-3-iodobenzene, CuI, K₂CO₃, DMEDA, DMF, 110 °C; and (e) R-prop-2-yn-1-ol, Pd(PPh₃)₂Cl₂, TEA/DMSO (2:1), 70 °C.

Scheme 3. Synthesis of the 4-aminopyrazolo[3,4-d]pyrimidine derivatives with a solvent-accessible moiety. Reagents and conditions: (a) CuI, Pd(PPh₃)₄, TEA, DMF, 70 °C; (b) Pd/C, H₂, MeOH, room temperature; (c) 1-bromo-3-iodobenzene, CuI, K₂CO₃, DMEDA, DMF, 110 °C; and (d) 1-Ethynylcyclohexanol, Pd(PPh₃)₂Cl₂, TEA/DMSO (2:1), 70 °C.

Table 2. PAK4 enzyme inhibitory activity and calculated properties of synthesised compounds

	PAK4 enzyme inhibition
Compound	% inhibitory activity	IC50 (μM)^b	clogP
10a	32	ND	3.79
10b	54	34.0	5.10
10c	29	ND	2.66
14a	60	ND	1.25
14b	97	0.77	2.57
14c	36	ND	0.12
14d	65	ND	2.59
18a	88	9.92	2.57
18b	90	4.41	3.78

ND: not determined

^aRelative percentage inhibition at 20 μM of compounds with staurosporine, HotSpot kinase assay (Reaction Biology Corp.).

^bHotSpot kinase assay (Reaction Biology Corp.).

Table 3. PAK subtype selectivity of SPA7012

Subtype	% inhibitory activity^a	IC50 (µM)^b
PAK1	16	ND
PAK2	36	NA
PAK3	5	ND
PAK4	81	0.77
PAK5	52	>100
PAK6	36	NA

ND: not determined; NA: no activity

^aRelative percentage inhibition at 20 μM of compounds with staurosporine, HotSpot kinase assay (Reaction Biology Corp.).

^bHotSpot kinase assay (Reaction Biology Corp.).

Figure 2. Attenuation of hepatic I/R injury by SPA7012. (A) Schematic diagram of treatment of SPA7012 in C57BL/6 mice. (B) Serum levels of AST and ALT (n = 5). (C, D) Gross morphology of livers, microscopic pictures of liver sections and measurement of necrotic area (n = 5). (E) Immunofluorescence staining and quantification of TUNEL-positive apoptotic cells in liver tissues (n = 5). (F) Western blotting analysis of apoptosis-related proteins in liver tissues (n = 5). Values are the mean ± SD.** p < 0.01. I/R, ischaemia-reperfusion; HPF, high power field.

Figure 3. Attenuation of I/R-induced oxidative stress by SPA7012. (A and B) Hepatic levels of malondialdehyde (MDA) and glutathione (GSH) (n = 5). (C) Immunohistochemical staining for 4-hydroxynonenal (4-HNE) of liver tissues (n = 5). (D) A co-IP analysis in HEK293T cells after 24 h transfection. (E) Nrf2 protein levels in nuclear-(NE) and cytosolic extracts (CE) of I/R injured liver tissues. (F) ARE luciferase activity in HEK293T cells after 24 h transfection (n = 5). (G and H) Nrf2 and its target gene levels in liver tissues (n = 5). Values are the mean ± SD. **p < 0.01. I/R: ischaemia–reperfusion; HPF: high-power field; V: vehicle; S: SPA7012 50 mg/kg.

Figure 4. Attenuation of I/R-induced inflammation by SPA7012. (A) Immunofluorescence staining and quantification of F4/80-positive macrophages and Ly6G-positive neutrophils in liver tissues (n = 5). (B and C) The protein and mRNA levels of pro-inflammatory cytokines/chemokine in serum and liver tissues (n = 5). (D) Protein levels of NF-κB signalling pathway (n = 5). Values are the mean ± SD. **p < 0.01. HPF: high-power field.

Figure 5. Attenuation of H/R-induced apoptotic cell death and cytokine production by SPA7012. (A) Schematic diagram of treatment of SPA7012 to primary hepatocytes. (B) MTT assay for cell viability (n = 5). (C) Lactate dehydrogenase (LDH) release from primary hepatocytes after 12 h reoxygenation (n = 5). (D and E) Analysis of apoptotic cell death by Annexin-V staining and Western blotting (n = 5). (F and G) Reactive oxygen species levels assessed using fluorescence microscopy following the changes in DCF fluorescence and colorimetric analysis of MDA and GSH (n = 5). (H and I) The protein and mRNA levels of pro-inflammatory cytokines/chemokine in culture media and hepatocytes (n = 5). (J) Protein levels of NF-κB signalling pathway (n = 5). Values are the mean ± SD. *p < 0.05 and **p < 0.01. H/R: hypoxia-reoxygenation.