Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase

Figures & data

Figure 1. Structures of the studied compounds: (A) potential inhibitors with the covalent mode of action and (B) the non-covalent control analogues, derived from analogues with the non-covalent mechanism of action (C).

Table 2. Effects of Imidazo[1,2-a]pyridine Analogues of α-phosphonocarboxylates on HeLa Cell Viability^a and Rab11A prenylation^b

Compound	R	Reduction of HeLa cell viability (IC50/µM)^d	Inhibition of Rab11A prenylation (LED/µM)^g	Densitometry analyses of Rab11A in cytosolic fractions after treatment with 100 µM concentrations of compounds (% of control ± SD)^h
7^f	CH3	222^f	10^f	335 ± 69^f
1a		81	NE	–
1b		NE	NE	–
1c		549	NE	–
2a		513	NE	–
3a		528	NE	–
2b^e		154	25	608 ± 136
2b incubated^c		280	NE	–
3b		198	NE	–
3b incubated^c		265	NE	–
2c		NE	NE	–
3c		NE	NE	–
2d		NE	NE	–
3d		NE	NE	–
1d^e		755	50	440 ± 132
1e^e		996	25	244 ± 91
1f		NE	NE	–
2e^e		154	25	659 ± 122
3e		NE	NE	–

^aViability was measured after 72 h of incubation. Data represent the means from at least three independent experiments.

^bCells were treated for 48 h with 100 µM concentration of the compounds. Rab11A and β-actin were detected in cytosolic fraction using Western blot. Data, from at least three independent experiments, indicates which bands corresponding to Rab11A had higher intensity compared to non-treated control.

^cCells were treated with decomposition products of compounds 2b and 3b (PBS/D₂O, 37 °C, 72 h).

^d“NE” (not effective up to 2 mM concentration).

^eCompounds in bold were evaluated to inhibit Rab11A and Rap1A/Rap1B prenylation in a wide concentration range.

^fliterature values determined using the same protocol.¹⁷.

^gLED: lowest effective dose.”NE” (not effective at 100 µM concentration).

^hDensitometry analysis representing relative unprenylated protein band intensity normalised to β-actin and quantified with respect to untreated cells.

Table 3. Peptides identified in MS and intensity ratio of untreated vs treated with 2b inhibitor samples.

Cys position in RABGGTB	Peptides identified	Intensity ratio untreated vs 2b treated^a
40	GSKKDDYEYCMSEY	0.83 (1)
78 & 82	IKSCQHECGGVSASIGHDPHLLY IKSCQHECGGVSASIGHDPHLL	1.06 ± 0.03 (6)
148	RFSFCAV SFCAVATL	0.90 ± 0.04 (7)
174	VLSCMNFDGGF VLSCMNF	0.91 ± 0.33 (2)
183	GCRPGSESHAGQIY	0.99 ± 0.03 (3)
196 & 197	GQIYCCTGFLA CCTGFLAITSQL	256^b (2)
220	GWWLCERQLPSGGL WLCERQLPSGGL LCERQLPSGGL	0.87 ± 0.53 (8)
240	NGRPEKLPDVCY	1.03 ± 0.10 (4)
270	ILACQDEETGGFADRPGDMVDPFHTLF ILACQDEETGGFADRPGDMVDPF ILACQDEETGGF	0.92 ± 0.06 (6)
314	SLLGEEQIKPVSPVFCMPEEVL CMPEEVL	0.93 ± 0.06 (5)

^aAverage value calculated based on data obtained from 8 LC-MS/MS measurements for the peptides listed ± StD, the number of individual ratios measured is given in parentheses; ^bSignal of peptides from the 2b treated samples was gone completely (in such cases, ratio = 256 is automatically assigned by the PEAKS software).

Scheme 1. Strategies used for the synthesis of α-phosphonocarboxylates 1. Reagents and conditions: (a) NaBH₄, NiCl₂·6H₂O, MeOH; (b) for 12a,c and 14d,f (n = 0): N,N-dimethylacrylamide or ethyl acrylate, Pd(AcO)₂, tri-o-tolylphosphine, DIPEA, propionitrile, microwave irradiation; for 12b and 14e (n = 2): (I) allyl alcohol, Pd(AcO)₂, tri-o-tolylphosphine, DIPEA, propionitrile, microwave irradiation; (II) triethylphophonoacetate, NaH, THF; (c) NaH, NFSI, THF; (d) (I) bromotrimethylsilane, TEA, acetonitrile, (II) EtOH, (III) TFA.

Scheme 2. Strategies used for the synthesis of α-phosphonocarboxylates 2–3. Reagents and conditions: (a) (I) 8, H₂, Pd/C, (II) Boc₂O, DCM, (III) NaBH₄, NiCl₂·6H₂O, MeOH; (b) NaH, NFSI, THF; (c) for 2 b,c: (I) 3 M HCl/EtOH, (II) acyl halide, DCM; (d) (I) bromotrimethylsilane, TEA, acetonitrile, (II) EtOH, (III) TFA; (e) (I) 7, 2-allylisoindoline-1,3-dione, Pd(AcO)₂, tri-o-tolylphosphine, DIPEA, propionitrile, microwave irradiation, (II) H₂, Pd/C, (III) NaBH₄, NiCl₂·6H₂O, MeOH; (f) (I) hydrazine hydrate, MeOH; (II) acyl halide or carboxylic acid with coupling agent in DCM or DMF; For details of synthesis of 2a,d, 3a see Schemes S7, S9.

Scheme 3. Strategies used for the synthesis of α-phosphonocarboxylates 4–6. Reagents and conditions: (a) Scheme S10–14; (b) tert-butyl 2-(diethoxyphosphoryl)acrylate, NaH, THF, then NFSI, THF; (c) (I) bromotrimethylsilane, TEA, acetonitrile, (II) EtOH, (III) TFA; (d) (I) 23, phthalic anhydride, AcOH, (II) Ph₃Cl, TEA, (III) hydrazine hydrate, MeOH, (IV) acyl halide, DCM, (V) TFA, DCM.

Table 1. Effect of exchanging electrophilic residue on half-Life Values ​​t_1/2 for reaction with GSH.

compound	R^a	t1/2^b
1a		40 h
1b		15 h
1c		stable^e
2a^c		0.18 h
2b		<0.25 h^d
2c		0.4 h
2d		0.97 h
3a		3.25 h
3b		<0.25 h^d
3c		56 h
3d		stable^e

^ain italic are indicated protons, which signals in the ¹H NMR spectrum were used for estimation of the reaction’s progress; ^bObtained values were calculated by fitting to a pseudo-first-order kinetic equation: tested compound integration = a * exp (-k * time); where a and k - constants of a given reaction³¹; ^creaction progress based on increasing signal of reaction product with glutathione; ^dobserved complete conversion of the test compound in the first ¹H NMR spectrum performed (after 15 min of reaction with GSH); ^eno reaction during 11 h of incubation.

Figure 2. Effect of imidazo[1,2-a]pyridine (1–3; A) and imidazole (4–6; B) PC analogues on Rab11A prenylation in HeLa cells. Cells were treated for 48 h with PCs (100 µM). Rab11A and β-actin were detected in cytosolic fraction using Western blot.

Figure 3. Effect of 6-substituted 3-IPEHPC analogue with the 3-ethoxy-3-oxopropyl group (1d), its version with an elongated carbon linker (n = 2; 1e), bromoacetamide analogue (2b) and its non-covalent counterpart (2e) on Rab11A and Rap1A/Rap1B prenylation in HeLa cells. Cells were treated for 48 h with the indicated concentrations of PCs (µM) or 10 µM lovastatin (Lov), acting as a positive control. Cytosolic fractions containing unprenylated proteins were Western blotted for Rab11A, Rap1A/Rap1B, and β-actin (A), and the bands were quantified by densitometry, normalised to β-actin, and presented as a percentage of controls (B). Data represented mean ± SEM from at least three independent experiments, *p ≤ 0.05, ****p ≤ 0.0001.

Figure 4. Effect of decomposition products of 2b and 3b on Rab11A prenylation in HeLa cells. Cells were treated for 48 h with 100 µM of freshly prepared 2b and 3b (-) and their solutions after incubation in PBS/D₂O at 37 °C for 72 h (+). Cytosolic fractions containing unprenylated proteins were Western blotted for Rab11A and β-actin.

Figure 5. Possible covalent bond formation by compound 2b suggested by docking simulations using 4GTS structure. 2b adopts a similar binding mode as the non-covalent compound 7¹⁷ (A). In not covalently bound state R144B stabilises 2b into the proximity of C196B (B) before the formation of the covalent interaction (C). The black dashed lines indicate interactions between the inhibitor and the protein. Black arrows indicate the formation of covalent bond. Used atom colours: C in protein amino acids, 7, 2b in unbound state, and 2b in bound state are grey, orange, light green, and light blue, respectively. O = red, N = blue, P = pink, S = yellow, F = dark green, Zn = golden, Br = magenta.

Kaźmierczak A, Kusy D, Niinivehmas SP, et al. Identification of the privileged position in the imidazo[1,2-a]pyridine ring of phosphonocarboxylates for development of Rab Geranylgeranyl Transferase (RGGT) inhibitors. J Med Chem 2017;60:8781–800.

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