Diabetes Drug Discovery: hIAPP1–37 Polymorphic Amyloid Structures as Novel Therapeutic Targets

Human islet amyloid peptide (hIAPP1–37) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1–37) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1–37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1–37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1–37. Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP1–37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A–F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities

Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures

<div><p>The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, <i>in silico</i> and <i>in vitro</i>, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones <b>A</b>, <b>B</b>, <b>C</b>, and <b>D</b>, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are able to protect and recondition the cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP_20–29 fragment or by a low potassium medium, regardless of their capacity for accelerating or inhibiting <i>in vitro</i> formation of fibers. <i>In vivo</i> animal experiments are required to study the impact of chemical chaperones in cognitive and metabolic syndromes.</p></div

Cell Apoptosis of CGC exposed to hIAPP_20–29, in monomeric and aggregated form, with and without N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B and H used as reference; at different molar ratio hIAPP_20–29:chaperones.

<p>In all assays caspase-3 levels were measured by immunofluorescence.</p

Cell Apoptosis of CGC exposed to pro-apoptotic stimulus as low potassium concentration (K5) during 4 h and later, the chaperones N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B and C (reference), were inoculated.

<p>In all assays caspase-3 levels were measured by inmunofluorescence.</p

Meta-structure of pharmacophore.

<p>A structural alignment using Pymol, whereby the interaction zones between β-amiloyd_17–42 (Aβ_17–42), IAPP and Eisenberg’s pharmacophore molecules and the chaperons were set in position with the interaction zone between BSA and the chaperons. BSA is shown in white at 70% transparency, the interaction zone between the Aβ_17–42 and the chaperons is shown in red, the one corresponding to the Eisenberg pharmacophore in blue, with IAPP in yellow and with BSA in green.</p

Values of molar ellipticity differences of the BSA fibrilogenesis assays at 70°C, in absence and presence of chaperones A, D, E, B, F and C, calculated from the CD spectra.

<p>*Differences of molar ellipticity at 240 and zero min. of incubation time.</p><p>Values of molar ellipticity differences of the BSA fibrilogenesis assays at 70°C, in absence and presence of chaperones A, D, E, B, F and C, calculated from the CD spectra.</p

Proposal mechanism of chaperone on protein aggregation/disaggregation.

<p>Novel chemical chaperones family can regulate fiber formation processes by binding to the native state, minimizing the formation of amorphous aggregates, as well as cytotoxic oligomers.</p

Transmission Electron Microscope (TEM) images of the BSA fibril formed with or without the chaperones (A, B, C, D, E, F and G) at a molar relation of BSA: chaperones 1:1, in buffer Tris (pH = 7.4, 20 mM) and 75°C.

<p>Scale bar 200 nm.</p

Visualization of binding site of chaperones (A, B, C, D, E, F, G and H) into IIA and IIIA regions of BSA, within 5Å distance.

<p>Visualization of binding site of chaperones (A, B, C, D, E, F, G and H) into IIA and IIIA regions of BSA, within 5Å distance.</p

ThT fluorescence kinetics during amyloid fibrillation of IAPP_20–29 (100 μmol/L).

<p>Experiments were carried out at 25°C in PBS buffer (pH 7.4; 10 mmol/L; NaCl 100 mmol/L), in presence or absence of the selected chaperones at molar relation 1:1 of IAPP_20–29: Chap. (ThT: 24 μM). Time dependent changes in ThT intensity was fitted by sigmoidal function (solid line). The experiments were carried out in triplicate. t_lag of fibril formation of the tested chaperones * p < 0,05 show significant differences with regard to control.</p