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Inhibitors of T3SS translocon assembly to combat multi-drug resistant P. aeruginosa

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R41AI149922-01
Agency Tracking Number: R41AI149922
Amount: $599,449.00
Phase: Phase I
Program: STTR
Solicitation Topic Code: NIAID
Solicitation Number: PA18-575
Solicitation Year: 2018
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-08-01
Award End Date (Contract End Date): 2022-07-31
Small Business Information
Worcester, MA 01605-4307
United States
DUNS: 158864715
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (508) 757-2800
Business Contact
Phone: (508) 757-2800
Research Institution
HADLEY, MA 01035-9450
United States

 Nonprofit College or University

Pseudomonas aeruginosa (PA) is the bacterium most frequently isolated from the respiratory tract of ICU pa-
tients in the US and is a major cause of pneumonia in intubated patients. Furthermore, infection with PA gener-
ally has a poor prognosis, with an estimated 40-69% of PA ventilator-associated pneumonia (VAP) cases re-
sulting in mortality, and more than 30% of survivors suffer recurrence following standard-of-care antimicrobial
therapy. The overall goal of this project is to address this critical medical need by discovering novel small mole-
cule inhibitors of the type-three secretion system (T3SS) translocon comprised of PopB andamp; PopD in the mam-
malian cell membrane and developing them into new therapeutic agents against PA. The T3SS is the major
virulence factor contributing to the establishment and dissemination of PA infections, and its presence is asso-
ciated with poor clinical outcomes and death in infected patients. The strategy of this project is to administer
T3SS inhibitors to PA pneumonia patients as adjunctive agents to enhance the function of standard-of-care
antibiotics by enabling phagocytic cells to eliminate persisters and antibiotic-resistant bacteria. Such agents will
by-pass the pathogen intrinsic resistance mechanisms -- a poorly permeable outer membrane and multiple ef-
flux pumps. The approach of this proposal is to identify small molecule inhibitors of the PopB/PopD translocon
assembly in cell membranes. In preliminary studies, the following was demonstrated: (a) PopB and PopD form
a hetero-dimer in model lipid bilayers, (b) PopB assists the insertion of PopD into the membrane of cultured
cells, and (c) only functional hetero-oligomers expose the N-terminus of PopD to the host cytosol. Conse-
quently, exposure of the PopD N-terminus to the host cytosol will be used as a reporter of accurate T3SS
translocon assembly. Self-complementation of a split green fluorescent protein (GFP) will be used to detect
properly inserted PopD. A truncated optimized superfolder-GFP (GFP1-10) missing a 16 amino acid residue β-
strand (GFP11), will be stably expressed in host mammalian cells. The missing GFP11 strand has been added
to the N-terminus of PopD as an epitope label. Assembly of functional translocons will expose the GFP11
epitope on PopD to the host cell cytosol where it will complement GFP1-10 and be detected by fluorescence.
Our studies have already demonstrated that GFP11-PopD is active for effector translocation when added to a
PA∆popD strain. In Phase I, we will develop this fluorescent cellular HTS assay for inhibitors of T3SS trans-
locon assembly. Compounds will be screened and hits confirmed and validated with secondary assays and
counter-screens. Compounds that meet the assay funnel criteria will be prioritized for chemical optimization
and in vivo proof efficacy in Phase II. Specific Aims are: (1) Complete development of cellular screens to iden-
tify inhibitors of the T3SS translocon assembly (Yr 1); (2) Optimize the T3SS translocon assembly screen for
HTS, apply it to libraries of diverse compounds, and confirm inhibitors (Yr 1-2); (3) Validate T3SS translocon
assembly inhibitors to determine potency, selectivity, preliminary SAR, and predicted ADME properties (Yr 2).Narrative
The pathogen Pseudomonas aeruginosa is classified at the serious threat level by the CDC because nearly
30% of clinical isolates are resistant to three classes of antibiotics. This project aims to develop a new class of
anti-pseudomonal agents that act on a novel bacterial target, a secretion system that attacks human cells nor-
mally fighting the infection. In this project, a novel screen will be applied to identify inhibitors, which will be pri-
oritized by potency, safety, and drug-like properties for further drug development. These new drugs will act by
a novel mechanism of enhancing the host innate immune response to infection.

* Information listed above is at the time of submission. *

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