BACE-1 Inhibitors Alzheimer’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 Human β-Secretase (BACE1) Inhibitors: JMC 2010 (Wyeth) 4/13/11 1 CHEM E-120
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BACE-1 Inhibitors Alzheimer’s Disease Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical.
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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
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Insoluble, neurotoxic, protein clusters formed from Amyloid Precursor Protein (APP)
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 Screening
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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
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 Screening
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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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A-siteneeds 2 HBD
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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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Can extend from S1 into S3?
40IC50 = 605 nMBUT brain penetrationIC50 = 27 nM
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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)