Rapid detection of drugs for protein misfolding diseases.

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Rapid detection of drugs for protein misfolding
diseases.

• Alexey Krasnoslobodtsev,
• Department of Pharmaceutical Sciences,
• University of Nebraska Medical Center
• COBRE 16 January, 2009

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Protein misfolding and Diseases.

• Protein mislfoding and aggregation is linked to
  many pathological diseases Alzheimer's,
  Parkinsons, Huntingtons and prion diseases.
• These diseases have common molecular mechanisms
  including protein aggregation and inclusion body
  formation.

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Protein misfolding diseases.

• The current status of drug development for
  protein misfolding diseases
• Most studied Alzheimer's and Parkinsons
• Alzheimers 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.o
  rg/)
• Parkinsons 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.

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Possible therapeutic interventions for protein
misfolding diseases
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

• Skovronovsky D.M., et al., 2006, Annu. Rev.
  Pathol. Dis., 1151-70

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Projects 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.

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Specific Aims

• 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.

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

• 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.

• 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.

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Simple model system Short peptide from Sup35
yeast prion protein.
Sup35 is a translation termination factor.
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.
We have observed that ionic strength of solution
has a strong effect on aggregation kinetics of
this peptide.
Ionic strength 11 mM 150 mM
Lag time 10.8 hours 7.1 hours
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Pathological mutations of a-synuclein.
Pathological interactions.
- a-synuclein is expressed in the brain, normally
unstructured and water soluble. - It aggregates
to form insoluble fibrils (Lewy bodies) found in
Parkinsons disease. - Accumulation of Lewy
bodies causes cell death which leads to selective
loss of neurons ? progressive motoric
dysfunction.
F 58 pN
Wild Type
F 75 pN
A single point mutation in a-synuclein - A53T has
been identified in familial early-onset
Parkinson's disease . Previous reports have
shown that mutant a-synuclein aggregates more
rapidly than wild-type protein.
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Specific Aims

• 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.

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AFM force spectroscopy -High throughput
screening machine for detecting efficient
therapeutic agents
Force Spectroscopy is a novel nanotool with
enormous potentials for the high-throughput
screening of Alzheimer's and Parkinsons 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.
Control
Drug 1
Drug 2
Drug 3
Force of intermolecular interactions

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

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

• 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.

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• 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.

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Beyond Force SpectroscopyDynamic Force
Spectroscopy (DFS)
Dynamic force spectroscopy (measuring forces at
various loading rates) reveals structural
energetic information in complex system.
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.
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 a-synuclein dimer pH 2.7 gt 4
sec pH 3.7 gt 1.3 sec pH 5.1 gt 0.3 sec
Yu, J., et al., J. Mol.Biol., 2008, Vol. 384,
992-1001.
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Summary

• 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.
• 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.
• 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.