Discovery and optimization of a novel spiropyrrolidine inhibitor of β-secretase (BACE1) through fragment-based drug design.

The aspartyl protease β-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aβ-peptide, a major component of plaques in the brains of Alzheimer's disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizer's proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.

[1]  D. Schlatter,et al.  Substrate and Inhibitor Profile of BACE (β-Secretase) and Comparison with Other Mammalian Aspartic Proteases* , 2002, The Journal of Biological Chemistry.

[2]  Lin Hong,et al.  Structural locations and functional roles of new subsites S5, S6, and S7 in memapsin 2 (beta-secretase). , 2005, Biochemistry.

[3]  Letter to the Editor: Backbone Resonance Assignments of the 45.3 kDa Catalytic Domain of Human BACE1 , 2004, Journal of biomolecular NMR.

[4]  Gianni Chessari,et al.  Application of fragment screening by X-ray crystallography to the discovery of aminopyridines as inhibitors of beta-secretase. , 2007, Journal of medicinal chemistry.

[5]  Michael Czarniecki,et al.  Application of fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design to identify novel microM leads for the development of nM BACE-1 (beta-site APP cleaving enzyme 1) inhibitors. , 2010, Journal of medicinal chemistry.

[6]  Bruno Martoglio,et al.  Aspartic proteases in drug discovery. , 2007, Current pharmaceutical design.

[7]  A. Berger,et al.  On the size of the active site in proteases. II. Carboxypeptidase-A. , 1967, Biochemical and biophysical research communications.

[8]  Gerhard Klebe,et al.  An old target revisited: two new privileged skeletons and an unexpected binding mode for HIV-protease inhibitors. , 2005, Angewandte Chemie.

[9]  Junya Qu,et al.  2-Amino-3,4-dihydroquinazolines as inhibitors of BACE-1 (beta-site APP cleaving enzyme): Use of structure based design to convert a micromolar hit into a nanomolar lead. , 2007, Journal of medicinal chemistry.

[10]  J. Hardy,et al.  Amyloid deposition as the central event in the aetiology of Alzheimer's disease. , 1991, Trends in pharmacological sciences.

[11]  Gianni Chessari,et al.  Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency. , 2007, Journal of medicinal chemistry.

[12]  Paul Zuck,et al.  Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-secretase. , 2008, Journal of medicinal chemistry.

[13]  Joseph B. Moon,et al.  Design, synthesis, and crystal structure of hydroxyethyl secondary amine-based peptidomimetic inhibitors of human beta-secretase. , 2007, Journal of medicinal chemistry.

[14]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[15]  L A Hansen,et al.  The importance of neuritic plaques and tangles to the development and evolution of AD , 2004, Neurology.

[16]  G. McGaughey,et al.  Discovery of pyrrolidine-based β-secretase inhibitors: lead advancement through conformational design for maintenance of ligand binding efficiency. , 2012, Bioorganic & medicinal chemistry letters.

[17]  A. Tomasselli,et al.  Design of potent inhibitors of human beta-secretase. Part 1. , 2007, Bioorganic & medicinal chemistry letters.

[18]  A. Berger,et al.  On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. , 1968, Biochemical and biophysical research communications.

[19]  L Hong,et al.  Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. , 2000, Science.

[20]  M. Wolfe.  The γ-Secretase Complex: Membrane-Embedded Proteolytic Ensemble , 2006 .

[21]  G. Klebe,et al.  Targeting the Open‐Flap Conformation of HIV‐1 Protease with Pyrrolidine‐Based Inhibitors , 2008, ChemMedChem.

[22]  James E Audia,et al.  Robust Central Reduction of Amyloid-β in Humans with an Orally Available, Non-Peptidic β-Secretase Inhibitor , 2011, The Journal of Neuroscience.

[23]  T. L. Blundell,et al.  Structural evidence for gene duplication in the evolution of the acid proteases , 1978, Nature.

[24]  Martin Stahl,et al.  Tyramine fragment binding to BACE-1. , 2008, Bioorganic & medicinal chemistry letters.

[25]  Y. Kiso,et al.  Recent progress in the drug discovery of non-peptidic BACE1 inhibitors , 2009, Expert opinion on drug discovery.

[26]  Rajiv Chopra,et al.  Acylguanidines as Small-Molecule β-Secretase Inhibitors , 2006 .

[27]  G. Klebe,et al.  Unexpected Novel Binding Mode of Pyrrolidine‐Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV Protease , 2006, ChemMedChem.

[28]  Bo Feng,et al.  In Vitro P-glycoprotein Assays to Predict the in Vivo Interactions of P-glycoprotein with Drugs in the Central Nervous System , 2008, Drug Metabolism and Disposition.

[29]  H. Pajouhesh,et al.  Medicinal chemical properties of successful central nervous system drugs , 2005, NeuroRX.

[30]  J. Pflugrath,et al.  The finer things in X-ray diffraction data collection. , 1999, Acta crystallographica. Section D, Biological crystallography.

[31]  A. Tomasselli,et al.  High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms. , 2008, Protein and peptide letters.

[32]  M Katharine Holloway,et al.  Identification of a small molecule nonpeptide active site beta-secretase inhibitor that displays a nontraditional binding mode for aspartyl proteases. , 2004, Journal of medicinal chemistry.

[33]  M. Malamas,et al.  Thiophene substituted acylguanidines as BACE1 inhibitors. , 2007, Bioorganic & medicinal chemistry letters.

[34]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[35]  Leighton Coates,et al.  X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases , 2006, European Biophysics Journal.

[36]  H. Sham,et al.  Improving the permeability of the hydroxyethylamine BACE-1 inhibitors: structure-activity relationship of P2' substituents. , 2010, Bioorganic & medicinal chemistry letters.

[37]  A. Hopkins,et al.  Ligand efficiency: a useful metric for lead selection. , 2004, Drug discovery today.

[38]  Gerhard Klebe,et al.  Structure-guided design of C2-symmetric HIV-1 protease inhibitors based on a pyrrolidine scaffold. , 2008, Journal of medicinal chemistry.

[39]  John-Michael Sauer,et al.  Design and synthesis of cell potent BACE-1 inhibitors: structure-activity relationship of P1' substituents. , 2009, Bioorganic & medicinal chemistry letters.

[40]  Rutger H A Folmer,et al.  Discovery of a novel warhead against beta-secretase through fragment-based lead generation. , 2007, Journal of medicinal chemistry.

[41]  Gianni Chessari,et al.  Application of fragment screening by X-ray crystallography to beta-secretase. , 2007, Journal of medicinal chemistry.

[42]  P. Verhoest,et al.  Defining desirable central nervous system drug space through the alignment of molecular properties, in vitro ADME, and safety attributes. , 2010, ACS chemical neuroscience.

[43]  M. Malamas,et al.  Acylguanidine inhibitors of β-secretase: Optimization of the pyrrole ring substituents extending into the S1 and S3 substrate binding pockets , 2008 .

[44]  Gianni Chessari,et al.  From fragment to clinical candidate--a historical perspective. , 2009, Drug discovery today.

[45]  D. Wyss,et al.  Competition STD NMR for the detection of high‐affinity ligands and NMR‐based screening , 2004, Magnetic resonance in chemistry : MRC.