3. • biological function of a protein depends on 3-D structure,
determined by its amino acid sequence during the process of
protein folding
• hallmark event in PCD is a change in the secondary and/or
tertiary structure of a normal protein
INTRODUCTION
4. Protein folding, misfolding and
aggregation
Protein aggregation
Chaperones
Protein fibrils
Environmental
Stress
Generic
Perturbations
Chemical
Stress
Pathophysiological
Stress
Disease
Native
folded
protein
Misfolded
protein
6. Protein-misfolding diseases
include conditions where a protein:
• fails to fold correctly (cystic fibrosis, Marfan syndrome,
amyotonic lateral sclerosis)
• is not stable enough to perform its normal function
(many forms of cancer)
• fails to be correctly trafficked (familial hypercholesterolemia,
α1-antitrypsin deficiency)
• forms insoluble aggregates that deposit toxically
(neurodegenerative diseases: Alzheimer’s, type II diabetes,
Parkinson’s and many more)
9. INTRABODY TECHNOLOGY
• gene-based strategy that relies on expression of recombinant
antibodies in different intracellular compartments to block or
modulate function of target molecules
• The single chain variable fragment (scFv) is the most popular
format for intrabodies
• Other formats that have been successfully expressed inside
cells are diabodies, single VL domains and single VH domains
Biochimica et Biophysica Acta 1792 (2009) 634–642
10. • variety of different conformers are the target antigens of the
intrabody approach against misfolding diseases
• isolation and characterization of these isoforms is a crucial
step for the development of conformation-specific intrabodies
for therapeutic purposes
INTRABODY TECHNOLOGY
Biochimica et Biophysica Acta 1792
(2009) 634–642
12. INTRABODY TECHNOLOGY
• antihtt intrabody C4
• intrabody EM48
HUNTINGTON
DISEASE
• inhibit the generation of the toxic amyloid beta-
peptide
• intrabodies directed to peptide adjacent to the
bsecretase cleavage site of human APP (scFv-b1)
ALZHEIMER’S
DISEASE
• anti-prion 8H4 monoclonal antibodies (mAbs)
• impairment of prion maturation
PRION
DISORDERS
13. CHAPERONE THERAPY
• principle of chaperone therapy is to restore the enzyme
activity by low molecular inhibitors with appropriate
molecular structure fitting in the enzyme molecule
• three types
a) chemical chaperones
b) non-competitive chaperones
c) molecular chaperone
14. Chemical Chaperones
• Active-site binding low molecular competitive inhibitors
• stabilized and enhanced the enzyme activity in somatic cells
by correcting the misfolding of enzyme protein.
• reached the brain through the blood-brain barrier after oral
administration, and correctedpathophysiology of the disease
15. • EXAMPLES
GLYCEROL,DMSO
ANTHRACYCLINES,PORPHY
RINS,DIAZO DYES
QUINACRINE
Chemical Chaperones
Nature 426, 905-909(18 December 2003)
16. • They are non-substrate-like compounds that exhibit allosteric
chaperone activities, not necessarily binding to the active site
of the enzyme.
• A set of pyrazolopyrimidines were identified by high
throughput screening as activators of O-glucosidase
responsible for Gaucher disease
• N-acetylcysteine (NAC) was reported to be a novel allosteric
chaperone for ,-glucosidase in Pompe disease. It improved the
stability and catalytic activity of recombinant ,-glucosidase as
a function of pH and temperature without disrupting its
catalytic activity. NAC does not interact with the catalytic
domain of the enzyme
Non inhibitory Chaperones
17. • utilize the heat shock protein and other chaperone proteins
induced by small molecules
• Major classes of Hsp are Hsp100, Hsp90, Hsp70, Hsp60 and
the small Hsp.Bind chemically unfolded polypeptides and
prevent aggregation
• A variety of molecular chaperones such as Hsp70 and Hsp40
have been demonstrated to exert therapeutic effects
• Hsp70 and its co-chaperone can arrest neurodegeneration by
preventing, α-synuclein misfolding, oligomerization and
aggregation in vitro and in Parkinson disease animal models.
• Hsp104 is able to resolve disordered protein aggregates and
cross β amyloid conformers. These chaperones have a
complementary effect and can be a target for therapeutic
intervention in Parkinson disease
Molecular Chaperones
20. • Low molecular weight compounds that selectively recognize the
functional proteins, such as receptor ligands or enzyme inhibitors,
and rescue conformational mutants
• Functions
Restore protein function
Eliminate aggregation or accumulation of misfolded proteins
• Due to stronger binding, pharmacological chaperones are effective
at much lower concentrations, so have reduced toxicity compared
to chemical chaperones. chemical chaperones non-selectively
stabilize proteins, including non target proteins, and facilitate their
folding ,whereas pharmacological chaperones target specific
proteins and restore folding
Pharmacological Chaperones
25. RNA SILENCING
Human Molecular Genetics,
2011, Vol. 20, Review Issue 1
• miRNAs consist of a class of short
22 nt RNAs derived from longer
processed dsRNA precursors.
• Induce translation repression by
one or more of the following
mechanisms: transcriptional
cleavage, blocking of ribosomal
function, deadenylation or
shunting of mRNAs to
transcriptionally inactive
cytoplasmic P bodies
26. • Targeting the expression of enzymes (e.g. BACE1)
required for the proteolytic processing of APP, or by
directly targeting the expression of APP
ALZHEIMER’S
DISEASE
• Targeting a-syn expression with RNAi
PARKINSON’S
DISEASE
• Allele-specific silencing using RNAi sequences
directed at single nucleotide polymorphisms (SNPs)
in linkage disequilibrium with CAG expanded alleles
POLY-Q
DISEASES
• Knockdown of PrP by RNAi and resultant
inhibition of PrPSc replication in cell culture
PRION
DISEASES
RNA SILENCING
27. GAIM TECHNOLOGY
• Novel approach based on a coat protein of bacteriophage
M13 that binds, disaggregates and prevents the formation of
multiple amyloid structures implicated in neurodegenerative
disease.
• GAIM selectively targets a broad spectrum of misfolded
proteins by recognizing a common amyloid conformation, the
product of misfolded protein aggregation in diseases such as
PD and Alzheimer’s disease (AD)
FUTURE PROSPECTS
28. • NeuroPhage Pharmaceuticals, Inc., announced that the
discovery, preclinical development and clinical trial approach
for its lead candidate, NPT088, recognizes and disrupts the
shape of misfolded proteins and target them for degradation
through the body's natural mechanisms
FUTURE PROSPECTS
www.neurophage.com
29. ANTI OXIDANTS
• Many PMD diseases are associated with the mechanisms of
ageing, such as the accumulation over time of active oxygen
and nitrogen species. There is evidence that this process can
be arrested somewhat by antioxidants, suggesting the
potential benefit of antioxidant-based regimens.
FUTURE PROSPECTS
30. CONCLUSION
• Misfolded proteins accumulate intracellularly and cause cell
damage,leading to diseases
• At present, there is no disease-modifying or curative therapy
to treat them despite the fact that many positive results have
been reported in preclinical work
• Studies on potential strategies with encouraging results argue
that better results could be achieved by improving
therapeutic agent delivery and long-term expression.
were highlighted in an oral session at the Alzheimer's Association International Conference (AAIC) taking place July 18-23, 2015, in Washington, D.C. NPT088 contains the general amyloid interaction motif (GAIM), which allows the compound to universally
This oxidative stress leads to protein damage in the cell resulting in their accumulation and eventually to cell death through apoptosis
