Islet amyloid-induced cell death and bilayer integrity loss share a molecular origin targetable with oligopyridylamide-based α-helical mimetics.

Islet amyloid polypeptide (IAPP) is a hormone cosecreted with insulin. IAPP proceeds through a series of conformational changes from random coil to ß-sheet via transient α-helical intermediates. An unknown subset of these events are associated with seemingly disparate gains of function, including catalysis of self-assembly, membrane penetration, loss of membrane integrity, mitochondrial localization, and finally, cytotoxicity, a central component of diabetic pathology. A series of small molecule, α-helical mimetics, oligopyridylamides, was previously shown to target the membrane-bound α-helical oligomeric intermediates of IAPP. In this study, we develop an improved, microwave-assisted synthesis of oligopyridylamides. A series of designed tripyridylamides demonstrate that lipid-catalyzed self-assembly of IAPP can be deliberately targeted. In addition, these molecules affect IAPP-induced leakage of synthetic liposomes and cellular toxicity in insulin-secreting cells. The tripyridylamides inhibit these processes with identical rank orders of effectiveness. This indicates a common molecular basis for the disparate set of observed effects of IAPP.

Small molecule screening in context: lipid-catalyzed amyloid formation.

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the ß-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemer's and Parkinson's) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid-catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.

A peptidomimetic approach to targeting pre-amyloidogenic states in type II diabetes.

Protein fiber formation is associated with diseases ranging from Alzheimer's to type II diabetes. For many systems, including islet amyloid polypeptide (IAPP) from type II diabetes, fibrillogenesis can be catalyzed by lipid bilayers. Paradoxically, amyloid fibers are beta sheet rich while membrane-stabilized states are alpha-helical. Here, a small molecule alpha helix mimetic, IS5, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP-induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative alpha-helical region of IAPP, revealing that alpha-helical states are on pathway to fiber formation. IAPP is not normally amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.

The interplay of catalysis and toxicity by amyloid intermediates on lipid bilayers: insights from type II diabetes.

The dynamics, energies, and structures governing protein folding are critical to biological function. Amyloidoses are a class of disease defined, in part, by the misfolding and aggregation of functional protein precursors into fibrillar states. Amyloid fibers contribute to the pathology of many diseases, including type II diabetes, Alzheimer's, and Parkinson's. In these disorders, amyloid fibers are present in affected tissues. However, it has become clear that intermediate states, rather than mature fibers, represent the cytotoxic species. In this review, we focus particularly on lipid bilayer-bound intermediates. Remarkably, the precursors of these fibers are intrinsically disordered, and yet catalysis of beta-sheet formation appears to be mediated by the stabilization of alpha-helical states. On the lipid bilayer, these intermediate species have been implicated as cytotoxic through elimination of ionic homeostasis. Recent advances are enabling insights at a molecular level that promise to provide meaningful targets for the development of therapeutics.

Amide inequivalence in the fibrillar assembly of islet amyloid polypeptide.

Amyloid fibers are aggregated, yet highly ordered, beta-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with alpha-helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide.

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.