Development of highly potent melanogenesis inhibitor by in vitro, in vivo and computational studies

Figures & data

Table 1 Inhibitory effects of amide derivatives 4a–e and 6a–e on mushroom tyrosinase and porcine pancreas elastase

Compounds	Tyrosinase activity IC50 ± SEM (µM)	Elastase activity IC50 ± SEM (µM)
4a	10.97±1.3	41.88±7.5
4b	17.98±2.5	81.85±15.2
4c	1.80±0.31	32.93±9.4
4d	10.96±1.8	24.31±5.7
4e	20.20±4.9	33.73±7.2
6a	0.78±0.09	5.01±1.8
6b	1.81±0.4	13.32±3.2
6c	1.41±0.1	23.32±4.2
6d	0.15±0.01	3.73±0.5
6e	16.71±3.1	35.80±8.2
Kojic acid	17.20±2.6	–
Oleanolic acid	–	1.69±0.05

Note: Values are expressed as IC₅₀ ± SEM.

Abbreviation: SEM, standard error of mean.

Table 2 Inhibitory effects of amide derivatives 4a–e and 6a–e on human tyrosinase (from melanoma cells) free radical scavenging

Compounds	Human tyrosinase % inhibition	Free radical scavenging % inhibition
4a	61.25±3.9	25.23±1.0
4b	53.83±5.3	1.70±0.5
4c	66.67±6.4	2.4±0.6
4d	56.85±6.5	91.78±2.0
4e	54.62±5.8	7.12±0.8
6a	82.35±8.5	1.39±0.5
6b	53.70±4.2	1.15±0.4
6c	55.83±5.7	8.51±1.0
6d	91.87±8.43	23.51±2.0
6e	57.40±4.92	7.50±1.0
Kojic acid	72.94±5.92	–
Oleanolic acid	–	95.60±1.0

Notes: For human tyrosinase, the concentration of all inhibitors and kojic acid was 50 µg/mL, and for free radical scavenging, the concentration of all inhibitors and vitamin C was 100 µg/mL. Values are expressed as mean ± standard error of mean.

Table 3 Kinetic parameters of mushroom tyrosinase for L-DOPA activity in the presence of different concentration of compounds 4c, 6a, 6b and 6d

Code	Dose (µM)	Vmax (ΔA/sec)	Km (mM)	Inhibition type	Inhibition	Ki (µM)	Ki′ (µM)
4c	0.0	5.090×10^−6	0.138	Non-competitive	–	0.188	–
	3.125	2.545×10^−6	0.138
	6.25	1.696×10^−6	0.138
	12.5	1.288×10^−6	0.138
6a	0.0	5.621×10^−6	0.112	Mixed inhibition	–	0.84	1.4
	0.3915	4.294×10^−6	0.126
	0.783	3.909×10^−6	0.136
6b	0.0	5.555×10^−7	0.089	Mixed inhibition	–	2.2	3.8
	0.453	4.819×10^−7	0.108
	0.965	3.947×10^−7	0.119
	1.813	3.067×10^−7	0.123
	3.626	2.592×10^−7	0.131
6d	0.0	1.161×10^−5	0.217	Non-competitive	Irreversible	0.217	–
	0.08	9.598×10^−6	0.217
	0.16	6.233×10^−6	0.217
	0.32	4.787×10^−6	0.217

Figure 1 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 4c.

Notes: (A) Concentrations of 4c were 0, 3.125, 6.25 and 12.5 µM and that of substrate l-DOPA were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope. The lines were drawn using linear least squares fit.

Figure 2 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 6a.

Notes: (A) Concentrations of 6a were 0, 0.3915, and 0.783 µM and those of substrate l-DOPA were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope and (C) of the vertical intercepts versus inhibitor 6a concentrations to determine inhibition constants. The lines were drawn using linear least squares fit.

Figure 3 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 6b.

Notes: (A) Concentrations of 6b were 0, 0.453, 0.965, 1.813 and 3.626 µM and those of substrate l-DOPA were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope and (C) of the vertical intercepts versus inhibitor 6b concentrations to determine inhibition constants. The lines were drawn using linear least squares fit.

Figure 4 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 6d.

Notes: (A) Concentrations of 6d were 0, 0.08, 0.16 and 0.32 µM and those of substrate l-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope. The lines were drawn using linear least squares fit.

Figure 5 Effect of various doses of mushroom tyrosinase on its activity for the catalysis of l-DOPA against different concentration of inhibitor 6d.

Figure 6 Effect of inhibitor 6d on pigmentation of zebrafish.

Notes: Embryos were treated with 5, 10, 20 and 50 µM of 6d and positive control kojic acid. (A) Representation of the pigmentation levels of zebrafish treated with inhibitor 6d and kojic acid. (B) Pixels comparison of the depigmenting potency of 6d and kojic acid. *P<0.05; **P<0.01.

Figure 7 Inhibitory effects of 6d and kojic acid on melanin contents.

Notes: Zebrafish embryos were treated with 50 µM of 6d and kojic acid. After homogenation and centrifugation, pellets were prepared and dissolved with 1 N NaOH at 100°C, and absorbances were recodred at 405 nm and compared with synthetic melanin. Values are expressed as % of control. *P<0.05; ***P<0.001.

Figure 8 Zebrafish embryos (48 hpf) were treated with 10, 20 and 50 µM of 6d.

Notes: Normal development was observed after acridine orange staining. Some key features were labeled for reference. a, eyes; b, otic capsule; c, heart; d, yolk; e, melanocytes.
Abbreviation: hpf, hours post-fertilization.

Table 4 Chemo-informatics evaluation of the synthesized compounds

Properties	4a	4b	4c	4d	4e	6a	6b	6c	6d	6e
MW	327	327	343	343	343	337	353	353	369	371
HBA	4	4	5	5	5	3	4	4	5	3
HBD	2	2	3	3	3	1	2	2	3	1
Log P	2.8	3.8	3.4	3.4	3.5	4.8	4.4	4.5	4.1	4.5
PSA	61	61	77	76	78	42	59	60	77	42
MV	328	328	339	341	339	361	372	372	382	378
Drug score	1.09	1.11	1.41	1.30	0.85	0.35	0.42	0.68	0.69	0.83
RO5	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes

Abbreviations: MW, molecular weight (g/mol); HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; Log P, lipophilicity of partition coefficient; PSA, polar surface area; MV, molar volume (A³); RO5, rule of five.

Figure 9 Docking energies of the synthesized amide derivatives 4a–e and 6a–e calculated using PyRx.

Figure 10 Docking interactions between 6d and target protein.

Notes: (A) The 6d docking complex with ligand is in blue color and embedded functional groups such as oxygen and amino are highlighted in red and dark blue colors, respectively. The protein structure is represented in purple color while interior helices are depicted in brown color. (B) Six copper-interacted residues lie within the active region of target protein and are represented in purple color. (C) The binding pocket of target protein in surface format is represented in dark purple color with conformational position of ligand. (D) Closer view of docking interaction. The active binding site amino acids are highlighted in brown color. Two copper ions are also represented in gray color. Two hydrogen bonds and one π–π interaction were observed between 6d and receptor amino acids such as His244, Met280 and His363 with bonding distances 1.98, 2.87, and 4.07Å, respectively. Interacted residues are in red color labels.

Figure 11 RMSD graph of 6a and 6d at 15 ns.

Note: The graph lines with purple and green colors represent 6a and 6d complexes, respectively.
Abbreviation: RMSD, root mean square deviation.

Figure 12 RMSF graph of 6a and 6d at 15 ns.

Note: The graph lines with purple and green colors represent 6a and 6d complexes, respectively.
Abbreviation: RMSF, root mean square fluctuation.

Figure 13 Radius of gyration (Rg) graphs of 6a and 6d: bound and unbound formats.

Note: The graph lines with green and purple represent the bound and unbound target protein, respectively.

Scheme 1 Synthesis of amide derivatives (4a–e).

Abbreviation: DMF, dimethylformamide.

Scheme 2 Synthesis of amide derivatives (6a–e).

Abbreviation: DMF, dimethylformamide.