BACE-1 InhibitorsAlzheimer’s Disease
Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and
Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1( β-Site APP Cleaving Enzyme 1) Inhibitors: JMC 2010 (Schering-Plough)
Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010 (Evotec)
Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I;Inhibitor Design and Validation: JMC 2010 (Schering-Plough)
Design and Synthesis of 5,5’-Disubstituted Aminohydantoins as Potent and Selective Humanβ-Secretase (BACE1) Inhibitors: JMC 2010 (Wyeth)
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Amyloid Plaque Formation
2
Insoluble, neurotoxic, protein clusters formed from Amyloid Precursor Protein (APP)
770 AA AA669 Asp
BACE β-secretase
AA669AA770
Β-CFT
γ-secretase
Aβ – β-amyloid1-40(42)
AChEfibril
peripheral siteof AChE
N terminusextracellular
C terminusintracellular
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BACE β-secretase
Transmembrane protein : NH2-terminal protease domain, connecting region, transmembrane region, cytosol domain
Aspartic acid protease with a catalytic diad of 2 aspartic acid amino acids
BACE-1 cleaves APP in the interior of the cell at a pH of ~ 5
BACE patents filed 1997 – 2010 (C&EN 2010, April 5)
Amgen 5BMS 25Elan 65Eli Lilly 3GSK 15Merck 45Schering-Plough 25Pfizer 50Wyeth 18
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BACE β-secretase
Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1 ( β-Site APP Cleaving Enzyme 1) Inhibitors
JMC 2010, 53, 942-950 from Schering-Plough
Strategy:
Discover low molecular weight compounds by Nuclear Magnetic Resonance – binding affinity
Validate hits by X-ray cystallography
Determine IC50 of enzyme inhibition
Optimize hits by small focused libraries
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Fragment-Based Screening
Discover of a “lead” compound – a compound that binds to the target, has functional activity, and possesses “drug-like” properties.
Modern methods:
1. High-throughput screening of large numbers of compounds: 10,000’s to 100,00’s2. Fragment-Based lead discovery: screen “components” of drugs, <5000 compounds
Screen small compounds of molecular weights 120-300Rule of 3 mw < 300 HBA < 3 rotatable bonds < 3
HBD < 3 clogP < 3
Thought is these compounds are more likely to fit into a binding site, the binding affinity
is usually quite low – KD of 100 μM to 10 mM“hit-rate” of 2-10%Target structure known either by X-ray crystallography or NMRStart to combine this fragment with others to fit better into the binding site with
greater affinity.Structure-guided modification of the lead fragment.
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Fragment-Based Screening
Chemical Fragments that Hydrogen Bond to Asp, Glu, Arg, and His Side Chains in ProteinBinding Sites: J Med. Chem 2010, 533086
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Fragment-Based NMR Screening2D 15N-HSQC NMRMeasure the changes in chemical shifts of 15N-labled BACE-1 catalytic domain in the presence of a fragment.
Express BACE-1 with 15NH4Cl as sole nitrogen sourcePurifyAssign resonances - Identify the amino acids in the catalytic domain in the NMR spectrum
Crystal structure helpsAdd a mixture of 10 compounds and measure the spectrum, compare with spectrum of the active site without compoundsHit – measure each compound separately to identify hitQuantify by Kd
1/τ = koff + kon[S]y = b + mx
kobs (s-1)
[S]
koff (Kd)
Δδ (ppm) vs [drug]
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Heteronuclear Two-Dimensional Coupling
HMQC (heteronuclear multiple quantum correlation)HSQC (heteronuclear single quantum correlation)
Correlation between the carbon atoms and the hydrogens attached DIRECTLY to the carbon, 1H vs 13C
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Fragment-Based NMR ScreeningScreened 10,000 compound fragment library, ~ 50% obeyed Rule-of-3
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Fragment-Based NMR Screening
Selected 204 isothioureas from fragment library and IC50 for BACE-1 inhibition measured15 compounds active at 50 μg/mL which where characterized by NMR to generate 336 fold increase binding affinity
got X-Ray structure
Ligand Efficiency (LE) free binding energy per non-hydrogen atom (kcal/mole/heavy atom)LE > 0.3
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Fragment-Based NMR ScreeningIsothioureas will have hydrolytic stability problems - bioisosters
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Fragment-Based NMR ScreeningAnalyzed 32 2-aminopyridines by NMR giving compound 4
Kd = 32 μMLE = 0.38similar to 3but no inhibition of BACE-1 up to 1 mMbut pKa ~ 7.2good for BBB penetration
3
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Hit-to-Lead Optimization
Small-focused libraries by parallel synthesisstep e most likely
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Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec)
Screened a 20,000 compound fragment library, average mw = 250screened at 1 mM in typical enzyme assayhits confirmed using surface plasmon resonance (SPR) and X-ray crystallography
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Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec)
Able to cocrystallize 3 with BACE1Asp32&228 catalytic diad
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Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec)
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Fragment-based discovery and optimization of BACE1 inhibitors: BMCL 2010, 20, 5329 (Evotec)
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Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I;Inhibitor Design and Validation: JMC 2010, 53, 951-965
Problem – need to cross BBB (pKa’s of ~ 7 are best) but the enzyme exists at a pH ~ 5
They focused on the second heterocycle – bioisotere of thioisourea with pKa > 6-10
4 HBD in protonated state4/13/11 CHEM E-120
Test the idea
Computational ligand binding studies suggested the chlorophenyl group will fit into S1
Parallel Synthesis
CH3NH2 PhCH2NH2 XPhCH2NH2 RCH2NH2
P1A
P1B
P1C
P1D P4D
ROW A
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Type 2 did not bind by NMR and did not inhibit BACE-1
Type 1 hits
Discovered two modes of binding
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Problem! Have two binding modes that are not obvious to control. Know want to extend into S1, S3 and S1’. With what??
Prepare a library 500 compounds using high throughput organic synthesis. 3 sites or points of diversity.
Fix R1 and R2 = CH3 and isobutylsteroechemistry?? Use racemic R3 = 500 amines
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IC50 = 27 nM
3 with IC50 = 0.2 mMto39 with IC50 = 27 nM
7407 fold enhancment
clog p = 7.5mw = 545LE = 0.25modest bioavailability
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Design and Synthesis of 5,5’-Disubstituted Aminohydantoins as Potent and Selective Human β-Secretase (BACE1) Inhibitors: JMC 2010, 53, 1146 (Wyeth)
Discovered through FRET assay
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