Diabetes mellitus drug discovery: insights into targeting feline and human amylin with small molecules

Figures & data

Figure 1. Representative figure displaying the four differing positions in the 37 amino acid peptide strands of human (hIAPP) and feline (fIAPP) islet amyloid polypeptide.

Scheme 1. Preparation of diaryl urea derivatives; dichloromethane (DCM), room temperature (rt), 8–12 h. Ar₁ (R₁) is a placeholder for one of the three series: 2-aminofluorene, 4-morpholinoaniline, or 4-aminoindole. Ar₂ (R₂) refers to one of the following substituents: o-methylphenyl, o-methoxyphenyl, o-fluorophenyl, o-nitrophenyl, p-(trifluoromethyl)phenyl, o,p-dimethoxylphenyl, o-methylchloro, m-(trifluoromethyl)phenyl, or o,p-dimethoxy.

Table 1. Chemical structure, physicochemical descriptors, and the effect of the 2-aminofluorene series of compounds on hIAPP and fIAPP aggregation measured as fluorescence intensity using ThT assay.

ID#	Structure	MW	TPSA	cLogP	cLogD	LR5	hIAPP	fIAPP
1		318.3443	41.13	4.22	5.03	N	39.5 ± 3.3	41.4 ± 0.6
2		368.3518	41.13	4.94	5.76	N	59.3 ± 2.7	63.7 ± 1.4
3		368.3518	41.13	4.96	5.76	N	83.3 ± 2.5	70.9 ± 1.2
4		345.3514	86.95	3.42	4.82	Y	30.8 ± 31	25.5 ± 1.9
5		348.8255	41.13	4.35	5.47	N	31.8 ± 0.4	16.3 ± 3.2
6		314.3804	41.13	4.23	5.4	N	36.0 ± 1.9	61.0 ± 3.8
7		330.3798	50.36	3.84	4.73	Y	22.1 ± 2.6	24.0 ± 1.1
8		360.4058	59.59	3.88	4.57	Y	45.6 ± 0.8	45.8 ± 8.6

Notes: TPSA, Log P, and Log D were obtained with SwissADME or Chemaxon. The ThT (%) represent the maximum fluorescence intensity obtained at the plateau phase of the kinetics of fibrillization using 10 µM of IAPP. Compounds were tested at 100 µM (molar ratio 1:10). ThT % indicated are average with SEM. These results are representative of three independent experiments performed with technical triplicates. cLogD: calculated distribution coefficient at 7.4; cLogP: calculated partition coefficient; LR5: Lipinski’s rule of five; MW: molecular weight; ThT: Thioflavin T; TPSA: topological polar surface area.

Table 2. Chemical structure, physicochemical descriptors, and the effect of the 4-morpholinoaniline series of compounds on hIAPP and fIAPP aggregation measured as fluorescence intensity using ThT assay.

ID#	Structure	MW	TPSA	cLogP	cLogD	LR5	hIAPP	fIAPP
9		315.3421	53.60	2.57	3.15	Y	42.6 ± 2.7	72.1 ± 1.8
10		365.3496	53.60	3.29	3.89	Y	15.1 ± 0.7	33.4 ± 4.3
11		365.3496	53.60	3.27	3.89	Y	13.4 ± 0.3	20.9 ± 0.6
12		342.3492	99.42	1.68	2.95	Y	9.8 ± 0.3	15.4 ± 0.4
13		345.8233	53.60	2.75	3.6	Y	20.7 ± 0.5	27.8 ± 3.3
14		311.3782	53.60	2.61	3.52	Y	31.5 ± 1.1	88.8 ± 3.4
15		327.3776	62.83	2.19	2.85	Y	13.6 ± 1.3	27.9 ± 1.0
16		357.4036	72.06	2.22	2.69	Y	27.2 ± 2.0	50.4 ± 1.6

Notes: TPSA, Log P, and Log D were obtained with SwissADME or Chemaxon. The ThT (%) represent the maximum fluorescence intensity obtained at the plateau phase of the kinetics of fibrillization using 10 µM of IAPP. Compounds were tested at 100 µM (molar ratio 1:10). ThT % indicated are average with SEM. These results are representative of three independent experiments performed with technical triplicates. cLogD: calculated distribution coefficient at 7.4; cLogP: calculated partition coefficient; LR5: Lipinski’s rule of five; MW: molecular weight; ThT: Thioflavin T; TPSA: topological polar surface area.

Table 3. Chemical structure, physicochemical descriptors, and the effect of the 4-aminoindole series of compounds on hIAPP and fIAPP aggregation measured as fluorescence intensity using ThT assay.

ID#	Structure	MW	TPSA	cLogP	cLogD	LR5	hIAPP	fIAPP
17		269.2737	56.92	2.88	3.36	Y	22.5 ± 0.1	25.3 ± 1.9
18		319.2812	56.92	3.58	4.1	Y	14.0 ± 0.3	15.0 ± 2.1
19		319.2812	56.92	3.60	4.1	Y	16.7 ± 0.3	23.2 ± 0.9
20		296.2808	102.74	2.09	3.16	Y	26.9 ± 0.3	44.5 ± 1.9
21		299.7549	56.92	3.11	3.80	Y	31.1 ± 0.2	39.9 ± 0.9
22		265.3098	56.92	2.90	3.73	Y	22.6 ± 1.0	34.1 ± 2.8
23		281.3092	66.15	2.54	3.06	Y	11.6 ± 0.2	18.1 ± 0.6
24		311.3352	75.38	2.54	2.90	Y	7.5 ± 0.4	6.9 ± 1.4

Notes: TPSA, Log P, and Log D were obtained with SwissADME or Chemaxon. The ThT (%) represent the maximum fluorescence intensity obtained at the plateau phase of the kinetics of fibrillization using 10 µM of IAPP. Compounds were tested at 100 µM (molar ratio 1:10). ThT % indicated are average with SEM. These results are representative of three independent experiments performed with technical triplicates. cLogD: calculated distribution coefficient at 7.4; cLogP: calculated partition coefficient; LR5: Lipinski’s rule of five; MW: molecular weight; ThT: Thioflavin T; TPSA: topological polar surface area.

Figure 2. Kinetics of IAPP fibril formation obtained with different diaryl derivatives of urea monitored with the thioflavin T (ThT) fluorescence assays. In this first-tier assay, compounds were tested at a final concentration of 100 μM in the presence of hIAPP and fIAPP at 10 μM. Molar ratio peptide: compound is 1:10. Series 1, 2, and 3 represent the 2-aminofluorene, morpholino, and 4-aminoindole, respectively.

Figure 3. Kinetics of IAPP fibril formation with increasing doses of three synthesized compounds 12, 23, and 24, assessed via thioflavin T (ThT) fluorescence assay. The original stock of the compound was tested at 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 µM concentration. These compounds were tested in the presence of hIAPP and fIAPP at 10 μM.

Figure 4. Size of hIAPP based on light scattering. Control at 60 min showed one major fibril peak in the 1000 nm range. The two tested compounds (12 and 24) were individually added to aliquots of hIAPP and incubated for 60 min before analysis to determine the size of the particles contained within the sample.

Figure 5. TEM imaging of amylin fibrils incubated with two unique treatments. Samples of fIAPP were solubilized at 10 µM in PBS buffer. Samples were incubated with DMSO (0.25%), compound 12, or compound 24. After the end of fibril formation kinetics (≤ 12 h), samples were deposited on copper grids and stained for TEM visualization. All images are displayed at 20k magnification.