# Development of LspA Inhibitors to Treat Gram-negative Bacterial Infections

> **NIH NIH R21** · PROKARYOTICS, INC. · 2022 · $170,000

## Abstract

Recently, WHO and CDC designated Carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 ‘critical
superbug’ and an ‘Urgent Threat’, and warned that new treatments for superbugs, which kill nearly 50,000
Americans and Europeans a year, are unlikely to be developed in time if left to market forces alone. Few
therapeutic options are left to treat CRE, and the fear of ‘pan-resistant’ CRE has emerged. Currently, most CRE
infections occur in a hospital setting, but the potential spread of CRE in the community also exists. As current
treatments are administered intravenously in a hospital setting, future agents offering oral administration would
reduce hospital stay and overall healthcare costs. Entirely new agents with novel mechanisms of action languish;
therefore, mechanistically novel antibiotics unaffected by clinically relevant resistance mechanisms and suitable
for orally administered stepdown therapy are urgently needed. Our proposal aims to develop a mechanistically
novel, IV and PO administered agent to treat infections caused by antibiotic susceptible and multidrug-resistant
Enterobacteriaceae, including extended spectrum beta-lactamase producers and CRE. Using an innovative
overexpression-based co-culture screen in Escherichia coli (Ec), we identified a small molecule inhibitor series
targeting LspA, the essential and broadly conserved Gram-negative lipoprotein signal peptidase that plays a
crucial role in outer membrane lipoprotein biogenesis. Preliminary work presented in this proposal establishes
the great potential of this series and target. We seek to continue to develop this program through the following
aims in a Hit-to-Lead campaign:
Aim 1 - Develop toolset to prosecute LspAi Hit-to-Lead campaign. (1) Synthesize 25 mg of LspAi screening
hits as well as key intermediates to interrogate nature of the warhead and facilitate analog synthesis in Aim 2.
(2) Test LspAis in Ec LspA in vitro biochemical assay to establish in vitro potency. (3) Expand FOR analysis in
Ec ΔtolC. (4) Perform FOR and mechanism of action (MOA) analysis in Klebsiella pneumoniae (Kp) ΔtolC to
show MOA extends to Kp.
Aim 2 – Hit-to-Lead campaign to identify 1 Lead series with WT activity and in vivo efficacy. (1) Hit-to-
Lead med chem analog synthesis. (2) MIC determination against WT and matched-pair efflux and permeability
modified Ec and Kp. (3) In vitro IC50 determination. (4) Assess plasma protein binding (ppb), mammalian HepG2
cytotoxicity, and hemolytic activity. (5) FOR determination, extended MOA analysis, kill curves and polymyxin
synergy assays. (6) Measure PK (IV, SC, and PO). (7) Compound scale-up for in vivo studies. (8) Conduct dose-
ranging mouse PK studies. (9) Demonstrate in vivo efficacy in murine septicemia model of WT Ec.
Aim 3 - Obtain Ec LspAi-LspA X-ray co-crystal structure in collaboration with SSGCID to enable
structure-based drug design approach. SSGCID-led Ec LspA heterologous expression, protein purification,
and crystallization studie...

## Key facts

- **NIH application ID:** 10508185
- **Project number:** 1R21AI171153-01
- **Recipient organization:** PROKARYOTICS, INC.
- **Principal Investigator:** Holly Ann Sutterlin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $170,000
- **Award type:** 1
- **Project period:** 2022-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10508185

## Citation

> US National Institutes of Health, RePORTER application 10508185, Development of LspA Inhibitors to Treat Gram-negative Bacterial Infections (1R21AI171153-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10508185. Licensed CC0.

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