Badger J.,Zenobia Therapeutics, Inc
Methods in Molecular Biology | Year: 2012
Crystallographic fragment screening is a technique for initiating drug discovery in which protein crystals are soaked or grown with high concentrations of small molecule compounds (typically MW 110-250 Da) chosen to represent fragments of potential drugs. Specific binding of these compounds to the protein is subsequently visualized in electron density maps obtained from analysis of X-ray diffraction data collected from these crystals. Theoretical and practical experience indicate that a suitably diverse library of fragment compounds containing only a few hundred compounds may be sufficient to provide a comprehensive screen of the protein target. By soaking crystals in mixtures of 3-10 compounds a fragment screen may be completed within ∼100 diffraction data sets. This data collection requirement may be met given reproducible well-diffracting protein crystals and robotic sample handling equipment at a high flux X-ray source. The leading practical issue for most crystallography laboratories that wish to launch a fragment screening project is the design and/or procurement of an appropriate fragment library. Although several off-the-shelf fragment libraries are available from chemical suppliers, the numbers, sizes, and solubility of the compounds in relatively few of these libraries are well-match to the specific needs of the crystallographic screening experiment. Informed consideration of the properties of compounds in the screening library, possibly augmented by additional filtering of available compounds with appropriate search tools, is required to design a successful experiment. The analysis of results from crystallographic fragment screening involves highly repetitive application of routine image data processing and structure refinement calculations from many very similar crystals. Efficient handling of the data applies a high-throughput structure determination methodology that conveniently packages the structure solution calculations into a single process that provides the crystallographer-analyst with ready-to-view maps for evaluating crystals for bound compounds. © 2012 Springer Science+Business Media, LLC.
Recht M.I.,Palo Alto Research Center PARC |
Nienaber V.,Zenobia Therapeutics, Inc |
Torres F.E.,Palo Alto Research Center PARC
Methods in Enzymology | Year: 2016
Isothermal titration calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of binding reactions. In addition, it enables label-free measurement of enzymatic reactions. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit validation and characterization, but its use in primary screening is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional ITC, particularly for screening libraries of 500-1000 compounds such as those encountered in fragment-based lead discovery. This chapter describes how nanocalorimetry and conventional microcalorimetry can be used to screen compound libraries for enzyme inhibitors. © 2016 Elsevier Inc. All rights reserved.
Nienaber V.L.,Zenobia Therapeutics, Inc
ACS Symposium Series | Year: 2011
Although diseases of the central nervous system are among the most devastating to patients and their families, disease modifying treatments have lagged behind other therapeutic areas. Current treatments were primarily discovered by serendipity and address disease symptoms. In the genomic era, understanding of CNS biology and disease associated mutations is growing thereby identifying a new series of putative targets. As CNS biology matures, there is growing need for a discovery paradigm that addresses the unique needs of CNS therapeutics, namely the ability of compounds to cross the blood-brain-barrier. The physiochemical properties of CNS therapeutics have been identified based upon historic data and may be used to guide discovery efforts. One notable variable is that the compounds should be low molecular weight. In this chapter, we discuss the merits of fragment-based lead discovery and how it may be used to address the challenges of CNS drug discovery. We also summarize practical strategies for library design and screening. Finally, we summarize examples of how fragments may be optimized into lead compounds. © 2011 American Chemical Society.
Zenobia Therapeutics, Inc | Date: 2012-06-22
Provided herein are compounds that inhibit or partially inhibit the activity of leucine rich repeat kinases. Also provided herein are methods of treatment of CNS disorders comprising administration of inhibitors of leucine rich repeat kinases.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 347.75K | Year: 2016
DESCRIPTION provided by applicant Enzyme activators are significantly underrepresented as therapeutic agents versus enzyme inhibitors There are only about a dozen examples of activator discovery in the scientific literature The lack of activator therapeutics is not from a ack of targets for diverse unmet medical needs Diseases such as neurodegeneration cancer and type diabetes could all benefit from an activator therapeutic This application introduces a new platform HOT ROXS for discovery of therapeutics for this class of andquot non druggableandquot targets HOT ROXS addresses three of the common issues in activator discovery a library rich in activators a generally applicable assay directed at activator identification and structural characterization of the activators to drive medicinal chemistry optimization of the hits To date most activator discovery has been through high throughput screening HTS HTS libraries are typically composed of large complex molecules Probability calculations indicate that complex molecules are much less likely to bind to a target than a smaller simpler compound fragments of drugs Furthermore the ligand binding efficiency binding energy per atom is typically much lower for HTS hits versus fragment hits This confounds medicinal chemistry optimization and can lead to flat SAR Here activators are defined as compounds that bind directly to the target of interest and stabilize it in the active conformation In HOT ROXS potential fragment activators are ideally identified as compounds that stabilize the active conformation of the protein by a positive shift in protein melting temperature In cases where the active conformation cannot be screened the inactive conformation is screened and the effect of activators on the melting temperature characterized early in the program by parallel activity screens Protein structure for the activators is initially measured in solution using Wide Angle X ray Scattering WAXS WAXS provides the molecular envelope for the protein ligand complex and is very sensitive to conformational shifts Changes as small as loop shifts can be detected by this method WAXS is used as part of an iterative process with single crystal x ray diffraction The initial x ray structure maybe apo or a ligand complex is fit to the WAXS pattern and changes upon activator binding identified The x ray structure may be remodeled to fit the new WAXS pattern WAXS is also very sensitive to conformational uniformity which is also a key characteristic for protein crystallization Activators are thought to increase the flexibility of proteins which would make crystallization of the complex more challenging So the WAXS pattern also identifies and prioritizes complexes for high throughput co crystallization studies WAXS may also identify different protein conformation classes which may streamline the co crystallization process or potentially provide for soaking of activators into pre formed crystals HOT ROXS has been used to identify activators for a high priority Parkinsonandapos s disease target and the method will be further developed and refined using this model system PUBLIC HEALTH RELEVANCE HOT ROXS is a new platform to facilitate discovery of therapeutic agents that activate the target of interest rather than inhibit it The platform includs a chemical library method to identify activator leads and a method to determine their structure in complex with the protein target The method may also be applied to other target classes and is not dependent on the disease to be treated