NIAID-RFQ21-2082644 - Statement of Work 08172021.pdf

STATEMENT OF WORK (SOW)

X-ray crystallography of the Protein-Ligand interaction between TLR4 and Pro-tryptase

Background The central goal of the Translational Allergic Immunopathology Unit (TAIU) at the National Institute of Allergy and Infectious Diseases (NIAID) is to identify mechanisms and pathways critical to allergic hypersensitivity that can be targeted in order to conduct first in human clinical trials that prevent severe reactions and inflammation. In order to accomplish this translational research objective, the TAIU has active clinical and laboratory research programs, employing a model of bed-to-bench and back to the bedside.

The principal approach TAIU employs for identification of therapeutic targets is to identify and characterize inherited, or acquired, genetic disorders and traits that promote severe allergic inflammation and reactions. From those identified to date, one major areas of current focus is characterizing pathways leading to increased mast cell hyperactivity and anaphylaxis in individuals with hereditary alpha tryptasemia (HaT) – a common genetic trait resulting from increased TPSAB1 (tryptase) copy number.

Scope The scope of this requirement is to solve the Protein-Ligand structures of TLR4 and Pro-tryptases.

Technical Requirements The Contractor shall:

• Develop a milestone plan in solving protein-ligand structures

• Design suitable protein constructs, based on its variety of TLR4 structures and NIAID’s experience with pro-tryptases.

• Screen constructs for expression in insect cells and shall establish purification protocols to produce Protein in purity and yields viable to support crystallization.

• Evaluate crystallization of selected constructs in a broad crystallization screen in presence and absence of ligands

• Further optimize detected protein crystals to yield a protein-ligand structure at a resolution of ≤3.0A

• At the request of NIAID Program staff, additional Ligand bundles for a regular protein-ligand structure analysis may be requested once a suitable crystallization system has been established

• Due to the nature of the interaction of these proteins, the Contractor shall perform capillary-free mounting of the crystals.

• Generate proteins/ligands in sufficient quality, quantity, and purity to solve the crystal structure

• Deliver one protein-ligand structure from each bundle of ligands provided by the Government

Other Requirements

• Protein purity standards must be at, or above, the requirement for crystallization.

• In order to facilitate success of this project, the contractor shall perform capillary-free mounting of the crystals.

Government Furnished Materials and Property For each Protein-Ligand-structure to be solved, the Government will provide a bundle of three (3) to five (5) Ligands in quantities and within the specifications needed. The contractor shall then deliver one Protein-Ligand structure from each bundle of ligands. Ligands provided by the Government shall exhibit the following properties:

- Solubility not less than 2 μM in aqueous buffer systems at neutral pH

- Ratio of Solubility / Kd should be ≥ 10.

Additionally, the Government will provide the following information on Ligands:

- binding/inhibition constant (Kd or Ki, or if unavailable IC50)

- molecular weight

- chemical structure, if required

Reporting Requirements and Deliverables After achievement of the milestones, the Contractor shall deliver a report generated under the Project describing the respective structures and activities including the electronic files as outlined below. These reports shall be provided at approximately week 13, 24, 27, and 30.

- Protein-Ligand.pdb

- Protein-Ligand.cv

- Protein-Ligand.mtz

- Protein-Ligand_2Fo-Fc.ccp4

- Protein-Ligand_Fo-Fc.ccp4

These reports shall document all interaction observed between TLR4/LPS/pro-tryptase and appropriate images of structures and interactions of those structures.

Following successful establishment of the crystal structure, the Contractor shall provide the protein used for crystallization to the Government.

Inspection and Acceptance Final inspection and acceptance of all work, requirements and deliverables shall be performed by Program officials, Jonathan Lyons and Michael O’Connell. Reports shall be complete and accurate in order for acceptance. Contractor shall only invoice once inspection and acceptance has been conducted by Program officials.

Period of Performance Period of performance shall be from date of award through 30 weeks.