Download Brandeis University Jeffery W. Kelly The Scripps Research Institute

Adapting the Chemistry and/or Biology of
Protein Homeostasis to Ameliorate
Aggregation-associated Diseases
2012 Leonard Berg Symposium
September 28, 2012
Washington University School of Medicine
Jeffery W. Kelly
The Central Dogma of Molecular Biology
DNA
RNA
Protein
Function
Protein Folding can be Spontaneous in vivo, However, for the
Majority of the Proteome, Folding is Biologically Assisted
Biologically
Assisted Folding
– Spontaneous Peptidyl-prolyl amide bond isomerization is too slow to support life
– Spontaneous Disulfide bond formation is also too slow to support life
Constant Competition Between Folding, Misfolding, Aggregation
Misfolding
Folding
Aggregation
Protein Homeostasis is Maintained by the
Proteostasis Network (>2500 Proteins)
quality
control
chaperones
transport
components
proteasome
autophagy
Balch, W.E. et al Science 2008, 319, 916.
The Cellular Proteostasis Network is
Regulated by Stress-responsive Signaling Pathways
Stress Sensor
Stress Sensor
Stress Sensor
Stress Sensor
Two Types of Aggregation-prone Proteins
Transthyretin, Lysoszyme, β2-microglobulin, Light Chain–all Natively
Folded Proteins Associated with Amyloid diseases
Hurle, M.R.; Helms, L.R.; Li, L.; Chan,
W.; Wetzel, R. “A role for destabilizing
amino acid replacements in light-chain
amyloidosis” Proc. Natl Acad. Sci,
1994, 91, 5446-5450
Colon, W.; Kelly, J.W. "Partial
Denaturation of Transthyretin is
Sufficient for Amyloid Fibril Formation
In Vitro." Biochemistry, 1992, 31,
8654-8660
Lai, Z.; Colon, W. ; Kelly, J.W. "The
Acid-Mediated Denaturation Pathway of
Transthyretin Yields A Conformational
Intermediate Which Can Self-Assemble
Into Amyloid" Biochemistry, 1996, 35,
6470-6482…….
Aggregation
Booth, D.R.; Sunde, M. Bellotti, V.;
Robinson, C.V.; Hutchinson, W.L.;
Fraser, P.E. Hawkins, P.N.; Dobson,
C.M. Radford, S.E.; Blake, C.C.F;
Pepys, M.B.; “Instability, unfolding and
aggregation of human lysozyme variants
underlying amyloid fibrillogenesis”
Nature, 1997, 385, 787-793;
Not All Aggregation-prone Proteins Can Fold!
Proteostasis of the Intrinsically Disordered Proteome ???
Conformational Change Hypothesis Still Applies
Aβ linked to Alzheimer’s, α-synuclein associated with Parkinson’s, and
exon 1 of the Huntingtin protein connected to Huntington’s disease are all
intrinsically disordered proteins
Aggregation
Ameliorating Protein-Misfolding/Aggregation
Diseases–A Systems Approach via Stressresponsive Signaling Pathway Activation
One drug → many diseases
Perform Trial in a Tractable
Disease, for intrinsically
disordered & Foldable
Proteins
Balch, W.E. et al Science 2008, 319, 916.
The Unfolded Protein Response Stress-responsive
Signaling Pathway–Secretory Pathway Proteostasis
Figure 2. The three arms of the unfolded protein response
Producing Arm-Selective, Constitutively Active Transcription Factors
in Organisms Allows One to Learn Which Arms Should Be Activated
to Ameliorate Specific Diseases
Genetically Encoded Small Molecule Regulated Destabilized
Domain Technology to Explore Arm-selective Unfolded
Protein Response (UPR) Transcriptional Programs
Banaszynski, L.A. et al Cell 2006, 126, 827.
Iwamoto, M. et al Chem. Biol. 2011, 18, 619.
Trimethoprim (TM)P)
stabilizes DHFR
In Collaboration w Luke Wiseman Assistant Professor of Mol. and Exp. Medicine
Research Conducted by Matthew Shoulders, now Asst. Prof. Chem. MIT
Pharmacologic Chaperone Regulated Destabilized Domain
Transcription Factors Exhibit Linear Dose Dependent Activation
Matthew Shoulders
DHFR-ATF6 and tet-Inducible XBP1s were cloned into into a
HEK293T Clonal Cell Line Refered to as HEK293DAX Cells
Matthew Shoulders
Matthew Shoulders and Lisa Ryno
The Transthyretin (TTR) Amyloidoses Result
From Post-secretion Misfolding & Aggregation
The Liver Secretes
Transthyretin
15
Protein Folding vs. Degradation in the
Endoplasmic Reticulum
Reticulum
• Network Regulation Strategy
can be used for both
foldable and intrinsically
disordered proteins
Proteasome-mediated
Degradation
Transcription Factor Regulation Identifies the Influence of
Arm-Selective Activation in Relevant Amyloid Cell Lines
ATF6 reduces secretion of
amyloidogenic transthyretin (TTR)
Amyloidogenic light chain
trafficking enhanced by XBP1s
and reduced by ATF6
Translating this to Small Molecule Arm-Selective Activators
of Transcription Factors Verify Luminescent Reporter
Constructs for Chemical Screens
Chemical Approaches for Ameliorating ProteinMisfolding/Aggregation Diseases
Balch, W.E. et al Science 2008, 319, 916.
The Transthyretin Amyloidoses Are in trans Gain-ofProteotoxicity Diseases Linked to Extracellular Misfolding
and Misassembly of Transthyretin
TTR Aggregation Destroys
Post-Mitotic Tissue!
Cytoplasmic Protein
The Transthyretin Amyloidoses
3-4
Transthyretin-Prominent Plasma Protein–Retinol Binding
Protein Carrier
≈ 50 %
≈ 50 %
127AA β-sheet rich 55 kDa homotetramer
Present in serum & cerebral spinal fluid
TTR-(RBP)2
Transthyretin (TTR)
Transport Thyroxine Retinol Binding Protein
In Humans Thyroxine Carrier Function Not Used
By Understanding The Transthyretin Aggregation Pathway
We Were Able to Conceive of a First-in-Class Drug
Rate Limiting
X
Biochemistry, 1992, 31, 8654-8660; Current Opinion In Structural Biology, 1996,
6, 11-17Nature-Strutural Biology, 2000, 7,754-757; Biochemistry 2001, 40,
11442-11452.
TTR Dissociation Pathway
Foss, et al. Biochemistry 2005 44, 15525-15533; Foss, et al. J. Mol. Biol. 2005 347, 841-854.
All TTR Disease Variants Are Destabilized
Biochemistry 1993, 32, 12119-12127, Biochemistry, 1995, 34, 13527-13536,
Proc. Natl. Acad. Sci. 2002, 99, 16427-16432, Cell 2005 121, 73-85.
The More Destabilized the Tetramer, the
Higher the Concentration of the
Amyloidogenic Monomer
Ligand Binding to TTR Can Prevent The Tetramer Amyloidogenic Intermediate Transition
Most Conservative Approach as it Does Not
Presuppose What the Toxic Species is !
Miroy et al. Proc. Natl. Acad. Sci.; 1996, 93, 15051-15056; Johnson et al. Acct. Chem. Res. 2005 38, 911-921.
A Key Human Genetics Observation From Dr. Teresa
Coelho Regarding Disease Etiology
• V30M FAP (polyneuropathy) highly penetrant in Portugal
• Family with V30M mutation that does not develop FAP
• These individuals also have a mutation, T119M, on their second
allele
• Hence the T119M TTR mutation appears to protect against
V30M amyloidogenesis in trans through mixed tetramer
formation
A T119M/V30M Compound Heterozygous Family Reveals That Interallelic
Trans–Suppression Ameliorates Pathology By Slowing Amyloidogenesis
Relative Fibril Formation
pH 4.4, 37 °C
3.6 µM TTR
Time (h)
V30M Disease Associated Subunits =
Hammarstrom et al.
Science 2001,
293, 2459-2461;
Science 2003,
299, 713-716.
T119M trans-suppressor subunits =
T119M Subunit Incorporation Increases the Dissociative Kinetic
Barrier Preventing Amyloidogenesis and Amyloidosis
Hammarstrom et al. Science 2001, 293, 2459-2461;Science 2003, 299, 713-716.
Activation Barrier Tuning w Small Molecules Mediated
by Native State Kinetic Stabilization
Activation Free Energy
Hammarstrom et al. Science 2003, 299, 713-716;
Razavi, Powers et al. Angew. Chem. Int. Ed. 2003, 42, 2758-2761;
Johnson. Acct. Chem. Res. 2005 38, 911-921.
TTR Kinetic Stabilizer From Structure-based Design
Tafamidis Kd1 = 2 nM, t1/2=20h
Bulawa, C.E.; Connelly, S.; DeVit, M.; Wang, L. Weigel,
C.;Fleming, J. Packman, J.; Powers, E.T.; Wiseman, R.L.; Foss,
T.R.; Wilson, I.A.; Kelly, J.W.; Labaudiniere, R. Proc. Natl. Acad.
The Tafamidis TTR Kinetic Stabilizer is Excellent at
Inhibiting TTR Amyloidogenesis
72 h
[Tafamidis] µM
Bulawa, C.E.; Connelly, S.; DeVit, M.; Wang, L. Weigel, C.;Fleming, J. Packman, J.; Powers, E.T.; Wiseman,
R.L.; Foss, T.R.; Wilson, I.A.; Kelly, J.W.; Labaudiniere, R. Proc. Natl. Acad. Sci. 2012 109, 9629-9634
NIS-LL Neurological Exam–Sensation, Muscle
Strength and Lower Limb Reflexes-Change From
Dr. Richard Labaudiniare
Baseline
Dr. Teresa Coelho et al.
Dr. Donna Grogan
Mr. Jeff Packman
20 mg once daily
Σ7 NTs nds (Large Fiber Function) Five Nerve
Conduction Measurements, Vibratory Threshold,
Heart Rate Response to Deep Breathing
20 mg once daily
Dr. Richard Labaudiniare
Dr. Teresa Coelho, et al.
Dr. Donna Grogan
Mr. Jeff Packman
mBMI-Modified Body Mass Index–Change From
Baseline–Improved Cachexia & Autonomic Neuropathy
Dr. Richard Labaudiniare
Dr. Teresa Coelho, et al.
Dr. Donna Grogan
Mr. Jeff Packman
20 mg once daily
Only Drug Altering the Underlying Amyloid Disease Etiology
First-in-Class Drug for a Human Amyloid Disease
NIH DK046335
1st Pharmacologic Evidence Supporting the Amyloid
What is Next ?
Uncovering the Mechanism of Action by Which
Protein Aggregation Causes the Degeneration of
Post-mitotic Tissue Upon Aging
Early Diagnosis is Also a Focus, as it is Key to
Maximizing the Efficacy of Tafamidis
Acknowledgments
• Dr. Matt Shoulders
• Ms. Lisa Ryno
• Dr. Luke Wiseman
• $$ NIH, NIDDK, NIGMS, NIA, Lita Annenberg Hazen
Foundation, Skaggs Institute of Chemical Biology
Acknowledgments–Part 1
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Dr. Ehud Cohen
Prof. Andy Dillin
Dr. Jan Bieschke
Dr. Tingwei Mu
Dr. Luke Wiseman
Dr. Steven Johnson
Prof. Evan Powers
Mr. Derrick Ong
Dr. Yoshiki Sekijima
Dr. Anu Sawkar
Ms. Amber Murray
Dr. Laura Segatori
Dr. Qinghai Zhang
Prof. Bill Balch
Prof. Joel Buxbaum
Dr. Deguo Du
Dr. Megan Wang
Prof. Eliezer Masliah
Prof. Michael Oldstone
Dr. Colleen Fearns
$$ NIH, NIDDK, NIGMS, NIA, Lita Annenberg Hazen
Foundation, Skaggs Institute of Chemical Biology
Acknowledgments–Part 2
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Dr. Per Hammarstrom
Dr. Steven Johnson
Dr. Hans Purkey
Dr. Wilfredo Colon
Dr. Luke Wiseman
Dr. Xin Jiang
Prof. Evan Powers
Dr. Vicki Lai
Dr. Yoshiki Sekijima
Dr. Greta Miroy
Dr. Sungwook Choi
Dr. Michael Petrassi
Prof. Ian Wilson
Dr. Stephen Connelly
Prof. Joel Buxbaum
Mr. Jeff Packman
Dr. Hossein Razavi
Dr. Donna Grogan
Dr. Ted Foss
Dr. Richard Labaudiniare
$$ NIH, NIDDK, NIGMS, NIA, Lita Annenberg Hazen
Foundation, Skaggs Institute of Chemical Biology