Different chemical proteomic approaches to identify the targets of lapatinib

Figures & data

Figure 1. Predicted binding of lapatinib analogues to the EGFR receptor site (docked to PDB: 1xkk) (1a, 1c, 1e) and corresponding schematic diagrams (1b, 1d, 1f).

Scheme 1. Synthetic approach to synthesised analogues of lapatinib. Reagents and conditions: (a) PS-CDI, HOBT, DIPEA, DCM, DMF, rt; (b) TFA, DCM, rt; (c) PS-CDI, HOBT, DIPEA, DCM, DMF, rt; (d) NaH, DMF, rt; (e) K₂CO₃, MeOH, rt.

Scheme 2. Synthesis of intermediate 5. Reagents and conditions: (a) thallium(III) trifluoroacetate, TFA, NaI, CF₃SO₃H, H₂O, 80 °C, 2h; (b) (trimethylsilyl)acetylene, CuI, TEA, PdCl₂(PPh₃)₂, THF, rt, on; (c) Ph₃P, CBr₄, DCM, rt, on.

Table 1. In vitro activity of lapatinib and its analogues as EGFR inhibitors.

Compound	Structure	IC50 (nM)
1		0.83
2		1.60
3		3.60
4		7.80

Figure 2. Silver-stained gels after interaction of purified recombinant EGFR with immobilised lapatinib derivatives 3 (a) and 2 (b). Matrices alone were used as negative controls (/). Purified recombinant EGFR was used as additional control.

Figure 3. Silver stained gels of bound proteins after incubation of 2 (a) and 3 (b) derivatives with A431 cell lysate.

Table 2. Identified proteins captured by 2, as identified using ESI-MS.

Band	Name	Accession no.
1	Epidermal growth factor receptor	P00533
2	Glyceraldehyde-3-phosphate dehydrogenase	P04406
3	Prohibitin 2	Q99623
4	Prohibitin	P35232
5, 7, 8	ND	ND^a
6	Keratin, type I cytoskeletal 10	P13645

^aND: not determined.

Table 3. Identified proteins captured by 3, as identified using ESI-MS.

Band	Name	Accession no.
1	Epidermal growth factor receptor	P00533
2, 3	ND	ND^a

^aND: not determined.

Table 4. Identified proteins captured by 4, as identified using ESI-MS.

	Name	Accession no.
1	Epidermal growth factor receptor	P00533
2	Fatty acid synthase	P49327
3	Protein disulphide isomerase	P07237
4	Peroxiredoxin 1	Q06830
5	Pyruvate kinase PKM	P14618
6	Histone H4	P62805
7	40S ribosomal protein S5	P46782

Table 5. Identified lapatinib bound proteins across different proteomics technologies.

	Name	Accession no.
1	Epidermal growth factor receptor	P00533
2	Protein disulphide isomerase	P07237
3	Peroxiredoxin 1	Q06830
4	Prohibitin	P35232
5	Prohibitin 2	Q99623
6	Glyceraldehyde-3-phosphate dehydrogenase	P04406
7	Fatty acid synthase	P49327
8	Pyruvate kinase PKM	P14618
9	Histone H4	P62805
10	40S ribosomal protein S5	P46782

Scheme 3. Synthesis of analogue 2. Reagents and conditions: (a) PS-CDI, HOBT, DIPEA, DCM, DMF, rt; (b) TFA, DCM, rt.

Scheme 4. Synthesis of analogue 3. Reagents and conditions: (a) PS-CDI, HOBT, DIPEA, DCM, DMF, rt.

Scheme 5. Synthesis of analogue 4. Reagents and conditions: (a) NaH, DMF, rt; (b) K₂CO₃, MeOH, rt.

Scheme 6. Synthesis of intermediate 5. Reagents and conditions: (a) thallium(III) trifluoroacetate, TFA, NaI, CF₃SO₃H, H₂O, 80 °C, 2h; (b) (trimethylsilyl)acetylene, CuI, TEA, PdCl₂(PPh₃)₂, THF, rt, on; (c) Ph₃P, CBr₄, DCM, rt, on.