Rapid detection of drugs for protein misfolding diseases.

Alexey Krasnoslobodtsev,Department of Pharmaceutical Sciences,

University of Nebraska Medical CenterCOBRE 16 January, 2009

Protein misfolding and Diseases. Protein mislfoding and aggregation is linked to many pathological

diseases – Alzheimer's, Parkinson’s, Huntington’s and prion diseases.

These diseases have common molecular mechanisms including protein aggregation and inclusion body formation.

Protein aggregation

Chaperones

Protein fibrils

EnvironmentalStress

GeneticPerturbations

ChemicalStress

Pathophysiological Stress

Disease

Unfolded (partially unfolded) protein

Native folded protein

Misfolded protein

Protein misfolding diseases.

The current status of drug development for protein misfolding diseases: Most studied Alzheimer's and Parkinson’s –

•Alzheimer’s: Because there is no cure, managing the disease usually involves medications to control symptoms, in combination with various non-drug strategies designed to ease the suffering of the person afflicted as well as his or her family and caregiver. (http://www.alzinfo.org/)

•Parkinson’s: At this time no treatment has been shown to slow or stop the progression of this disease. Instead, therapy is directed at treating the symptoms that are most bothersome to an individual with Parkinson's disease. (http://www.parkinson.org/)

•Universally fatal diseases.

Possible therapeutic interventions for protein misfolding diseases

Skovronovsky D.M., et al., 2006, Annu. Rev. Pathol. Dis., 1:151-70

An emerging therapeutic strategy for protein misfolding diseases: Small molecules that bind to specific regions of the misfolded protein and stabilize it.

Chemical (pharmacological) Chaperones

Project’s objective.

• There is a critical need to identify effective therapeutic agents to treat and/or prevent these devastating diseases.

• There is a critical need for exploration of novel more rapid and efficient methods to study the molecular mechanisms underlying protein misfolding.

• The objective of the project is to evaluate single molecule force spectroscopy as a novel method that would identify pathological conformations of a protein molecule (misfolded states), determine factors stabilizing interactions between misfolded proteins, improve the decision making process for potential drug candidates.

Specific Aims1) Specific aim #1: Confirm the efficacy of single molecule force spectroscopy for

identifying pathological interactions. - Our working hypothesis is that the strength of interactions between individual proteins measured with single molecule force spectroscopy is linked to pathology.

2) Specific aim #2: Evaluate the possibility of using Force Spectroscopy for high-throughput

screening of potential drugs that prevent aggregation. - Our working hypothesis is that the use of single molecule force spectroscopy will significantly increase the speed of decision making for drug candidates in protein misfolding diseases.

3) Specific aim #3: Identify stabilizing factors that drive self-assembly of proteins leading to smart drug design.- We will test the hypothesis that the subset of interactions that contribute the most to the stability of aggregated species comes from the main chain hydrogen bonding between proteins which explains structural similarities of aggregates formed by proteins diverse in sequence.

Force Spectroscopy – a nanotool for early detection of protein misfolded states.

• Advantages: Force spectroscopy operates at single molecule level and it is

capable of detecting and analyzing aggregation prone conformations very early in the disease development – at the step of dimer formation.

• The misfolded protein conformation that is aggregation prone is different from other protein conformations by their increased propensity to interact with each another leading to aggregation.

• Force spectroscopy can detect such propensity by measuring strength of interactions between individual molecules.

The strength of interactions between proteins in misfolded state is elevated.

Simple model system: Short peptide from Sup35 yeast prion protein.

A seven amino acid sequence within the N-terminal domain is responsible for the aggregation of the whole Sup35 protein. This short peptide aggregates with the formation of fibrils.

1 122 253 685

7GNNQQNY13

Ionic strength 11 mM 150 mM

Lag time 10.8 hours 7.1 hours

We have observed that ionic strength of solution has a strong effect on aggregation kinetics of this peptide.

Sup35 is a translation termination factor.

0 40 80 120 1600

10

20

30

40

50 F = 33 pN

Cou

nts

Force, pN0 40 80 120 160

0

10

20

30F = 60 pN

Cou

nts

Force, pN

Pathological mutations of α-synuclein.

0 40 80 120 1600

5

10

15

20

25

Mutant A53TC

ount

s

Force/pN

F = 58 pN

A53T

F = 75 pN

A single point mutation in α-synuclein - A53T has been identified in familial early-onset Parkinson's disease .

Previous reports have shown that mutant α-synuclein aggregates more rapidly than wild-type protein.

Wild Type

Pathological interactions.- α-synuclein is expressed in the brain, normally unstructured and water soluble. - It aggregates to form insoluble fibrils (Lewy bodies) found in Parkinson’s disease. - Accumulation of Lewy bodies causes cell death which leads to selective loss of neurons → progressive motoric dysfunction.

Specific Aims1) Specific aim #1: Determine the benefit of single molecule force spectroscopy for

tracking(identifying) pathological interactions. - Our working hypothesis is that the strength of interactions between individual proteins measured with single molecule force spectroscopy is linked to pathology.

2) Specific aim #2: Evaluate the possibility of using Force Spectroscopy for high-

throughput screening of potential drugs that prevent aggregation. - Our working hypothesis is that the use of single molecule force spectroscopy will significantly increase the speed of decision making for drug candidates in protein misfolding diseases.

3) Specific aim #3: Identify stabilizing factors that drive self-assembly of proteins leading to smart drug design.- We will test the hypothesis that the subset of interactions that contribute the most to the stability of aggregated species comes from the main chain hydrogen bonding between proteins which explains structural similarities of aggregates formed by proteins diverse in sequence.

AFM force spectroscopy -High throughput screening machine

for detecting efficient therapeutic agents

Drug #2 is the best candidate for the development of effective therapeutic agents

Force of intermolecular interactions

Drug #1

Drug #2

Drug #3

Control

Force Spectroscopy is a novel nanotool with enormous potentials for the high-throughput screening of Alzheimer's and Parkinson’s drug candidates.

The flow cell mode allows testing the candidate drugs at various conditions approaching to the physiological ones with automated exchange of buffers containing drugs of interest.

This method would allow for rapid screening of drug candidates created through combinatorial chemistry.

Specific Aims1) Specific aim #1: Determine the benefit of single molecule force spectroscopy for

tracking(identifying) pathological interactions. - Our working hypothesis is that the strength of interactions between individual proteins measured with single molecule force spectroscopy is linked to pathology.

2) Specific aim #2: Evaluate the possibility of using Force Spectroscopy for high-throughput

screening of potential drugs that prevent aggregation. - Our working hypothesis is that the use of single molecule force spectroscopy will significantly increase the speed of decision making for drug candidates in protein misfolding diseases.

3) Specific aim #3: Identify stabilizing factors that drive self-assembly of proteins leading to smart drug design.- We will test the hypothesis that the subset of interactions that contribute the most to the stability of aggregated species comes from the main chain hydrogen bonding between proteins which explains structural similarities of aggregates formed by proteins diverse in sequence.

We will use a combination of main-chain mutations which would remove backbone hydrogen bonding in the core sequences of aggregation and Dynamic Force Spectroscopy.

Beyond Force SpectroscopyDynamic Force Spectroscopy (DFS)

Using DFS one can obtain the off-rate constant of spontaneous dissociation of the complex. This further characterizes the early stages of the misfolded protein interactions leading to the formation of protein dimers.

Dynamic force spectroscopy (measuring forces at various loading rates) reveals structural energetic information in complex system.

Yu, J., et al., J. Mol.Biol., 2008, Vol. 384, 992-1001.

The lifetime of the dimers is one of the important properties quantitatively characterizing the stability of the very first intermediate state of protein aggregates.

The lifetimes for the α-synuclein dimer: pH 2.7 => 4 secpH 3.7 => 1.3 secpH 5.1 => 0.3 sec

Summary

1. The project capitalizes on the use of a novel concept that Force Spectroscopy provides an advanced detection of protein conformational changes (misfolding) with enhanced interprotein interactions critically involved in aggregation process.

2. This novel nanoprobing approach will help in overcoming the limitations of traditional ensemble based methods to detect and analyze misfolded states of proteins. Application of this approach has a potential for rapid screening, analysis and evaluation of potential inhibitors (drugs) of protein aggregation. It is expected to speed the decision making and ultimately shorten the overall time for drug development process.

3. The work proposed in this project is expected to elucidate the mechanism of initial stages of aggregation which will have a positive impact on the development of rational effective therapeutics.