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

Abstract

Background: Type 2 diabetes (T2D) is a health concern for both humans and cats, with cases rising over the past decade. Around 70% of patients from either species exhibit pancreatic aggregates of islet amyloid polypeptide (IAPP), a protein that proves toxic upon misfolding. These misfolded protein aggregates congregate in the islets of Langerhans of the pancreas, diminishing the capability of β-cells to produce insulin and further perpetuating disease.

Objective: Our team's drug discovery program is investigating newly synthesized compounds that could diminish aggregates of both human and feline IAPP, potentially disrupting the progression of T2D.

Material and methods: We prepared 24 compounds derived from diaryl urea, as ureas have previously demonstrated great potential at reducing accumulations of misfolded proteins. Biophysical methods were employed to analyze the anti-aggregation activity of these compounds at inhibiting and/or disrupting IAPP fibril formation in vitro.

Results: The results demonstrate that compounds 12 and 24 were most effective at reducing the fibrillization and aggregation of both human and feline IAPP. When compared with the control for each experiment, samples treated with either compound 12 or 24 exhibited fewer accumulations of amyloid-like fibrils.

Conclusion: Urea-based compounds, such as compounds 12 and 24, may prove crucial in future pre-clinical studies in the search for therapeutics for T2D.

Keywords: Amylin; cat; drug discovery; islet amyloid polypeptide; pancreatic amyloidosis; small molecule therapeutics; type 2 diabetes; veterinary medicine.

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.

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.