What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Abstract

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests offpathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include nonlinear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

Estimation of the lag time in a subsequent monomer addition model for fibril elongation

“…Note that we do not include the intermediate ''docked'' monomertemplate fibril complexes in the above expression, because they are short-lived. To model this expression, we substitute the numerical solution of the ordinary differential equation system, eqn (4)- (8), into the fibril concentration term and calculate the weighted sum of the observable species. The observable eqn (30) is validated by how accurately it can monitor an increase in fluorescence, which is a measure of the formation of fibrils as a function of time.…”

“…6 The equilibrium fibril size distribution can vary from strongly skewed distributions to broad distributions depending on a number of factors, including elapsed time, fragmentation effects, and aggregate merging. 7,8 Estimation of the rates of aggregation reactions will be important not only for identification and overall understanding of aggregation mechanisms, but also for developing pharmacological treatment and strategies for disease prevention.…”

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

“…where and are determined by the elementary steps that increase the mass of insoluble aggregates (Figure b). Sigmoidal progress curves comprising an initial lag phase followed by an exponential phase and a plateau phase are expected in the cases of slow primary nucleation, for which the values of are lower than . In turn, fast primary nucleation processes ( ) give rise to hyperbolic progress curves that are also observed during seeded aggregation assays (see section 2.).…”

“…However, deciphering the exact mode of action of aggregation inhibitors is not a trivial task on account of the possible pathways (Figure a) and multiple elementary steps (Figure b) that might be implicated. It may occur, for instance, that large amounts of stable soluble oligomers are formed without their presence being directly detected by amyloid‐specific probes such as thioflavin‐T (ThT) or by turbidimetric methods …”

“…In turn, fast primary nucleation processes ( ) give rise to hyperbolic progress curves that are also observed during seeded aggregation assays (see section 2.). Protein aggregation modulators can be screened based on kinetic signatures standing out from the standard behavior expected by Equation (2) . This is valid for both on‐ and off‐pathway inhibition as it is shown next for the case of amyloid fibril formation.…”