Slides presented at the University of Edinburgh in Jan 2014, based on our paper "A tale of two drug targets: the evolutionary history of BACE1 and BACE2" http://www.ncbi.nlm.nih.gov/pubmed/24381583
The beta amyloid (APP) cleaving enzyme (BACE1) has been a drug target for Alzheimer's Disease (AD) since 1999 with lead inhibitors now entering clinical trials. In 2011, the paralog, BACE2, became a new target for type II diabetes (T2DM) having been identified as a TMEM27 secretase regulating pancreatic β cell function. However, the normal roles of both enzymes are unclear. This study outlines their evolutionary history and new opportunities for functional genomics. We identified 30 homologs (UrBACEs) in basal phyla including Placozoans, Cnidarians, Choanoflagellates, Porifera, Echinoderms, Annelids, Mollusks and Ascidians (but not Ecdysozoans). UrBACEs are predominantly single copy, show 35–45% protein sequence identity with mammalian BACE1, are ~100 residues longer than cathepsin paralogs with an aspartyl protease domain flanked by a signal peptide and a C-terminal transmembrane domain. While multiple paralogs in Trichoplax and Monosiga pre-date the nervous system, duplication of the UrBACE in fish gave rise to BACE1 and BACE2 in the vertebrate lineage. The latter evolved more rapidly as the former maintained the emergent neuronal role. In mammals, Ka/Ks for BACE2 is higher than BACE1 but low ratios for both suggest purifying selection. The 5' exons show higher Ka/Ks than the catalytic section. Model organism genomes show the absence of certain BACE human substrates when the UrBACE is present. Experiments could thus reveal undiscovered substrates and roles. The human protease double-target status means that evolutionary trajectories and functional shifts associated with different substrates will have implications for the development of clinical candidates for both AD and T2DM. A rational basis for inhibition specificity ratios and assessing target-related side effects will be facilitated by a more complete picture of BACE1 and BACE2 functions informed by their evolutionary context.
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and Back
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://cdsouthan.blogspot.se/2014/01/a-tale-of-two-targets-bace1-and-bace2.html
http://www.guidetopharmacology.org/
cdsouthan@hotmail.com
Orchid ID 0000-0001-9580-0446
Twitter: http://twitter.com/#!/cdsouthan
Blog: http://cdsouthan.blogspot.com/
LinkedIN: http://www.linkedin.com/in/cdsouthan
Presentations: http://www.slideshare.net/cdsouthan
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3. Presentation Outline
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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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5. BACE1 Normal Function: Neuronal Pleiotropic
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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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6. BACE2 Normal Function : Peripheral Pleiotropic
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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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15. A Tale of three Paralogs (I)
BACE1, Chromosome 11
BACE2, Chromosome 21
CATE,
Chromosome 1
50% identity
27% identity
Protein sequence comparison
mRNA tissue distribution
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16. A Tale of three
Paralogs (II)
InterProScan outputs
CATE
BACE1
BACE2
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20. 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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21. Rates of Recent Evolution
Ka/Ks vs exon position for human/mouse/rat
BACE1&2
0.25
Ka/Ks
0.2
0.15
BACE1
0.1
BACE2
0.05
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Exon Exon Exon Exon Exon Exon Exon Exon Exon
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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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22. 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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23. Digging Deep: “BACE-like” Criteria
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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 Nterminal 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 Nterminal match.
Iterating trees with different parameterizations, including cathepsins as
out-groups, support the UrBACE grouping
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24. 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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26. Oily Tails: the Key to Secretase Function
BACE1 Spalmitoylati
on at 4 Cys
residues
BACE2
UrBACE
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27. Animal Lineages with no UrBACE
Demosponge
Non-orthologous replacement of UrBACE for pre- or post- neuronal
RIP-related secretase functions ?
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28. 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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29. 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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30. 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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31. From Evolution back to Drug Discovery
PMID:23128209
PMID:23128209
http://en.wikipedia.org/wiki/Model_organism
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32. Opportunities for Functional Genomics
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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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33. 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. neuregulinsparing)
• 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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34. Questions ?
(and you can try your own phylogenies at home)
http://figshare.com/articles/Supplementary_Data_for_Southan_Hancock_BACE_evolution_paper/855620
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