Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target

Figures & data

Figure 1. Malonate (1), 2-methylmalonate (2), 2,2-dichloromalonate (3), 1,2-cyclopropanedicarboxylate (4).

Figure 2. Superposition of malonate (cyan) and cis-1,2-cyclopropanedicarboxylate (green).

Table 1. Compounds synthesized and tested for SR inhibitory activity.

Compound	R1	R2	R3	R4	Docking Score	SR Conformation	Average IC50 (mM)	Average KD (mM)
4	COOH	COOH	H	H	−9.037	Closed	3.3 ± 0.4	0.90 ± 0.025
5	COOH	H	COOH	H	−9.231	Closed	n. a.	> 5.0
(±)6	COOH	COOEt	H	H	−6.266	Half-closed	n. a.	> 5.0
(±)7	COOH	COOiPr	H	H	−5.519	Half-closed	> 5.0	2.5 ± 0.5
8	COOH	COOH	CH3	CH3	−8.661	Closed	>5.0	> 5.0
(±)9	COOH	H	Ph	H	–	–	> 5.0	> 5.0
Malonate					−9.939	Closed	n.a.	9.4·10^−3 ± 0.4·10^−3

Docking scores here reported are those obtained in the first docking run (without considering water molecules). Compound 9 was purchased at a later stage; therefore, docking was not performed for it. Docking scores for compounds 4, 5, 8, and malonate are referred to the closed enzyme conformation; docking scores for 6 and 7 are relative to the intermediate conformation.

Table 2. Available SR crystal structures: structural information and water molecules solved in the binding site.

	3L6R	3L6B	3L6C - A	3L6C - B	2ZR8	3HMK chain A	3HMK chain B	1WTC	2ZPU	1V71
Ligand and structural  annotations	Malonate	Malonate	Malonate	Malonate	Ser	Apo	Apo	Apo	Alanine covalently bound to PLP	Apo
Organism	Human	Human	Rat	Rat	S. pombe	Rat	Rat	S. pombe	S. pombe	S. pombe
Resolution	1.7	1.5	2.2	2.2	2.2	2.1	2.1	1.9	1.9	1.7
W1 num.	389	372	365	–	391	410	415	403	370	365
B factor	14.32	12.37	26.37	–	14.65	45.04	21.61	14.53	12.12	17.14
W2 num.	461	403	355	345	–	–	409	–	–	506
B factor	15.11	12.03	19.31	35.81	–	–	21.61	–	–	22.8
W3 num.	469	436	392	–	–	–	–	614	371	560
B factor	19.5	14.98	38.52	–	--	–	–	41.96	13.5	42.49
W4 num.	658	351	x	–	–	–	–	–	–	469
B factor	33.56	23.04	xx	–	--	–	–	–	–	37.12
W5 num.	585	373	393	–	–	–	–	416	368	410
B factor	16	13.06	31.06	–	–	–	–	16.54	13.13	20.91
W6 num.	626	412	403	--	–	––	366	–	–	–
B factor	18.47	17.45	37.26	–	–	–	22.11	–	–	–
W7 num.	399	385	–	–	378	–	–	–	385	–
B factor	20.9	23.18	–	–	23.76	–	–	–	15.85	–
W8 num.	392	355	355	346	399	424	454	430	369	361
B factor	13.67	10.91	23.3	37.96	10.23	18.91	15.44	23.64	8.96	14.46

Each water molecule (W1–8) in the binding site of SR crystal structures is reported the corresponding residue number and the B factor.

Figure 3. Dissociation constant and half-maximal inhibitory concentration of compound 4. (A) Fluorescence emission spectra, upon excitation at 412 nm, of a solution containing 2.4 μM hSR, 50 mM TEA, 150 mM NaCl, 5 mM TCEP, 1 mM MgCl₂, 10% DMSO (v/v), pH 8.0, and increasing concentrations of compound 4, at 20 °C. (B) Dependence on the concentration of compound 4 of the fluorescence emission intensity at 500 nm upon excitation at 412 nm. The experimental points were fitted by a binding isotherm, yielding a K_D of 0.90 ± 0.03 mM. (C) Dependence on the concentration of compound 4 of hSR β-elimination activity, at 37 °C. Experiments were carried out in an assay solution containing 0.4 μM hSR, 50 mM TEA, 150 mM NaCl, 36 mM l-Ser, 5 mM DTT, 1 mM MgCl₂, 50 μM PLP, 2 mM ATP, 10% DMSO (v/v), pH 8.0, and variable concentrations of compound 4. Experimental data were fitted to the Equation (1), yielding an IC₅₀ value of 3.3 ± 0.4 mM.

Figure 4. (A) Docking pose of compound 4; (B) binding mode of compound 5: the carboxylic group is located in proximity of a water molecule, that was omitted during docking simulation; (C) pose of compound 8; (D) docking pose of compound 7.

Figure 5. Conserved water molecules in the closed structure of SR. (A) Crystal structure of 3L6B; malonate is depicted in sticks and lemon green, water molecules are reported in ball and sticks. (B) Hbond network between malonate, water molecules and protein residues in the active site of 3L6B crystal structure.

Table 3. Docking experiments performed using the same conditions reported in the paper by Vorlova et al. performed using Glide, PLANTS, and AutoDock.

	IC50 (mM)	Docking score Glide – 2nd run	Docking score PLANTS	Docking score AutoDock
Malonate	0.067 ± 0.001*	−10.437	−75.2163	−7.94
2,2-Dichloromalonate	0.057 ± 0.001*	−9.938	−56.2757	−5.17
2-Methylmalonate	1.85 ± 0.001*	−10.115	−69.9599	−7.84
Compound 4	3.3 ± 0.4	−10.441	−63.6435	−7.53
Compound 8	> 5	−9.043

*IC₅₀ values published in Vorlova et al.25

Figure 6. Surface representation of the binding site features map: in blue are reported the Hbond donor areas, in red the Hbond acceptor areas and in yellow the hydrophobic areas. (A) 3L6B crystal structure (closed enzyme conformation). (B) Human model built on the rat 3HMK crystal structure (open enzyme conformation).

Scheme 1. Reagents and conditions A: (a) for 4, H₂O, 2 h at reflux; for 6, EtOH, 2 h at reflux; for 7, iPrOH, 2 h at reflux; (b) 1.KOH, reflux, 2 h; 2.HCl 1N; (c) NaH, toluene, 0 °C to RT, 48–72 h; (d) KOH, THF/water (1:2) from 0 °C to 50 °C, 4 d. Yields and purification methods are reported in the Supplementary information.

Vorlová B, Nachtigallová D, Jirásková-Vaníčková J, et al. Malonate-based inhibitors of mammalian serine racemase: kinetic characterization and structure-based computational study. Eur J Med Chem 2015;89:189–97

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