Post on 10-May-2015
description
[1]
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and Back
17th January 2014, QMRI, University of Edinburgh
Christopher Southan, IUPHAR Database and Guide to PHARMACOLOGY Web Portal Group, Queen's Medical Research Institute, University of Edinburgh, UK
http://www.guidetopharmacology.org/cdsouthan@hotmail.comOrchid ID 0000-0001-9580-0446Twitter: http://twitter.com/#!/cdsouthanBlog: http://cdsouthan.blogspot.com/LinkedIN: http://www.linkedin.com/in/cdsouthan Presentations: http://www.slideshare.net/cdsouthan
http://cdsouthan.blogspot.se/2014/01/a-tale-of-two-targets-bace1-and-bace2.html
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Reference and Acknowledgment
PMID:24381583 (2013)
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Presentation Outline
• Introducing the BACE1 and BACE2 Aspartyl proteases• BACE1 as an Alzheimer’s disease (AD) target• BACE2 as a Type II Diabetes (T2D) target• Drug discovery > evolution - the three paralogs• The chordate/vetebrate BACE1/2 evolutionary trajectory• The ancestral UrBACE in basal animal phyla and oily tails• Where the either the UrBACE or certain substrates are missing• Corroboration by another paper • Evolution back to drug discovery – functional genomics• Evolution informs drug development
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Starting Points
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BACE1 Normal Function: Neuronal Pleiotropic
• Review articles collate ~13 non-amyloidogenic in vivo substrates• Suggested normal cleaved APP (P05067) ectodomain involvement nerve cells
and Aβ peptide dampening of neuronal hyperactivity• Voltage-gated sodium channel subunits (SCN4B, O60939) substrates for
regulation of Nav1 channel metabolism• Neuregulin(NRG1, Q022979) substrate for control of nerve cell myelination• Amyloid-like protein 2 (APP2, Q06481) substrate for ectodomain fragments• Pancreatic ectodomain shedding of broad set of β-cell-enriched substrates• KO-mice subtle neurochemical deficits and behavioural changes• Zebrafish KO substrates related to neurite outgrowth and axon guidance• Zebrafish KO shows peripheral hypomyelination
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BACE2 Normal Function : Peripheral Pleiotropic
• Similar to BACE1 in catalytic activity with a large open cleft• Balance of evidence against being an APP beta secretase in vivo• Broad tissue distribution attributable to different promoter usage to BACE1• KO mice “normal” • Neonatal mortality increase in mouse double-KO (Bace1-/-; Bace2-/-) • TMM27 (Q9HBJ8) secretase in mice and in human pancreatic β-cell membranes• Pancreatic ectodomain shedding of narrow set of β-cell substrates• Processes mouse pigment cell-specific Melanocyte Protein (Q60696)• Zebrafish KO melanocyte migration phenotype. • Zebrafish Double KO (Bace1-/-; Bace2-/-) viable does not enhance the single
mutant phenotypes, indicating non-redundant functions
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Incidence of Alzheimer's Disease (AD)(by millions vs age in USA)
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A-beta 1-42 (sp|P05067|672-713)
Regulated Intramembrane Proteolysis (RIP): APP Secretases as AD Targets
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4865
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BACE1 Inhibitor Phase I (discontinued)AZD3839, Ki 26.1 nM
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6931
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Incidence of Type 2 Diabetes (by millions world-wide)
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PMID:21907142 (2011)
BACE2 Target Validation: 10 years after BACE1
PMID:23506624 (2012)
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6540
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Global Drug
Discovery Output
for BACE1 (left) and
BACE1 (right)
Southan C, Varkonyi P, Boppana K, Jagarlapudi SA, et al. (2013) Tracking 20 Years of Compound-to-Target Output from Literature and Patents.http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077142
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Drug Discovery Output for BACE1 from UKPMC
http://cdsouthan.blogspot.se/2013/11/drug-target-time-tracking.html
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From Drug Discovery to Evolution
PMID:12904811 (2003)
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A Tale of three Paralogs (I)
BACE1, Chromosome 11
BACE2, Chromosome 21
CATE, Chromosome 1
50% identity
27% identity
mRNA tissue distributionProtein sequence comparison
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BACE2
BACE1
CATE
A Tale of three Paralogs (II)
InterProScan outputs
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Challenges in Finding Orthologs: Truncated ORFs from Genome Pipelines
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Many Ensembl Orthologs have Errors
80 orthologs of BACE1 in Ensembl % target ID (blue) vs. % query ID
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BACE in Chordates
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BACE in Chordates
• Ensembl has dense vertebrate coverage so we chose spaced sampling• Amphioxus outgroup consistent with protochordate 2R whole-genome
duplication followed by paralog persistence • Distinctly accelerated evolution of BACE2 (neofunctionalisation ?)• Inferred neuronal role for BAC1 but no data outside human, mouse, fish • Long branches are partial sequences• Birds group with turtles• Coelacanth groups with reptiles and tetrapods (not ray-finned fish)• Xenopus laevis tetraploidisation has maintained “double” paralogs• No evidence for pseudogenes or “dead” variants• Implication of common origin between nerve and pancreatic cells
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Rates of Recent Evolution
Ka/Ks vs exon position for human/mouse/rat BACE1&2
0
0.05
0.1
0.15
0.2
0.25
Exon1
Exon2
Exon3
Exon4
Exon5
Exon6
Exon7
Exon8
Exon9
Ka/
Ks BACE1
BACE2
• Ka/Ks reflects the level of purifying selection• Exon 1 is subject to weak selection while exons 4-6 are under
strong selection (catalytic site)• Ka/Ks for BACE2 is on average twice that for BACE1
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Discovering the UrBACE: Human BACE1 vs Monosiga (Choanoflagellate)
33% identity over 432 residues, 9% gaps, divergence time ~0.8 billion years
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Digging Deep: “BACE-like” Criteria • Reciprocal BLAST highest score usually against a BACE1• Mostly single copies (i.e. just one UrBACE)• Identity across the major part of the ORF < 35% without over-gapping• Cathepsin paralogs drop to ~ 25% identity over ~350 residues with
~20% gapping • Typically ~100 residues longer than cathepsins • N-terminal signal peptide and a C-terminal transmembrane (CTM) either
side of the protease domain • CTM is absent from cathepsin paralogs although they also have N-
terminal signal peptides • Presence of diagnostic PRINTS matches, including at least one of the
profiles for BACE, typically BACE1 • Cathepsins and BACE2 sequences consistently showed two matches to
the Prosite PS00141. UrBACEs showed only the single proximal N-terminal match.
• Iterating trees with different parameterizations, including cathepsins as out-groups, support the UrBACE grouping
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The UrBACE Evolutionary Trajectory
• After duplication from a cathepsin ancestor the emergence of the UrBACE protein sequence is distinct
• Cathepsin paralogs form a clear outgroup • “Shuffling-in” of the CTM is the defining post-duplication shift in cellular
location and function• Multiple divergent paralogs in basal phyla (2 in Monosiga, 3 in
Trichoplax) predate the nervous system• Found in cnidaria with only nerve nets• Long branch lengths mainly due to partial sequences• Major orders now represented by draft genome assemblies• Basal relationships of Eumetazoans still unresolved• Beyond limited EST coverage no tissue distribution or functional data
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UrBACEs and Cathepsin Paralogs
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Oily Tails: the Key to Secretase Function
BACE1 S-palmitoylation at 4 Cys residues
BACE2
UrBACE
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Animal Lineages with no UrBACE
Non-orthologous replacement of UrBACE for pre- or post- neuronal RIP-related secretase functions ?
Demosponge
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Detecting BACE1 and BACE2 substrate homologs:(note UrBACE + and APP -)
BACE1 (P56817), APP (P05067), NRG1 (Q02297), SCN2B (O60939), TMEM27 (Q9HBJ8), and PSEN1 (P49768) for comparison
X represents probable absence by low BLASTP score (score matrix in supplementary data)
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The BACE that probably isn’t (because it’s a cathepsin sequence)
Neurotoxic effects induced by the Drosophila amyloid-beta peptide suggest a conserved toxic function (PMID:19049874, 2009)• “We therefore propose that this fly enzyme represents an endogenous
fly β-secretase and now named it dBACE”
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Independent Corroboration
Asynchronous Evolutionary Origins of Aβ and BACE1 (PMID:24361992, Dec 2013)
“sequences homologous to Aβ are not found outside gnathostomes and the β cut site is only conserved within sarcopterygians. BACE1 enzymes, however, extend through basal chordates and as far as cnidaria. We then sought to determine whether BACE1 from a species that never evolved Aβ could proteolyze APP substrates which include Aβ. We demonstrate that BACE1 from a basal chordate is a functional ortholog that can liberate Aβ from full length human APP, indicating BACE1 activity evolved at least 360 million years before Aβ”
Chinese Hamster Ovary cells transfected with human APP
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From Evolution back to Drug Discovery
PMID:23128209
PMID:23128209
http://en.wikipedia.org/wiki/Model_organism
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Opportunities for Functional Genomics
• UrBACE experiments to illuminate contemporary BACE roles and networks
• Comparative expression data to give clues• KO or RNAi for protease or substrate ablation• Mutants for more subtle perturbations• Chemical perturbations with BACE1 and/or BACE2 inhibitors as probes• Substitution options (e.g. swap UrBACE for human BACE1)• Omics profiling for phenotypic read-out• Smaller gene repertoires > simpler result interpretation • UrBACE –ve and APP (like) +ve organisms as important controls• Could detect UrBACE function shifts (e.g. pre and post-nervous system)• Vertebrates will also show functional shifts for BACE1 and BACE1 (e.g.
melanocyte role)
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Evolution-informed Drug Development
• Clinical candidate inhibitors face first-in-class challenges • Reciprocal specificity screening of both paralogs (in ChEMBL_17 the
tested compound ratio for BACE1:BACE2 is 3999:563)• Specificity ratios (e.g. IC50s for BACE1:BACE2) will be chosen more
carefully and factor-in brain penetration • New substrates verified in model organisms can be assessed for human
side-effect liabilities and biomarkers• Substrate-specific inhibitor selectivity could be an option (e.g. neuregulin-
sparing)• Screen BACE1 leads in T2D models and BACE2 leads in AD models • Possible new lower organism phenotypic assays for BACE inhibitors, even
with UrBACE• Important to develop pathway perspectives on both paralogs
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Questions ?(and you can try your own phylogenies at home)
http://figshare.com/articles/Supplementary_Data_for_Southan_Hancock_BACE_evolution_paper/855620