Discovery of novel anti-infectives for gram negative pathogens
Inventors
Bulterys, Philip • Miller, Jeffery F. • DAMOISEAUX, ROBERT D. • French, Christopher Todd
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Assignees
University of California San Diego UCSD
University of California, San Diego (UCSD)The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
Publication Number
US-12357617-B2
Publication Date
2025-07-15
Expiration Date
2039-10-04
Abstract
Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 select pathogens that cause highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ˜220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a BSL-2 surrogate for these pathogens. 268 hits were identified, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm. In a fulminant murine model of respiratory melioidosis, treatment with a number of these agents was significantly more effective than ceftazidime, the current drug of choice, for improving patient survival and decreasing bacterial counts in major organs.
Core Innovation
Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are highly infectious Gram-negative bacteria that cause diseases melioidosis and glanders, respectively, characterized by high lethality and limited therapeutic options. These pathogens display intrinsic and acquired antibiotic resistance mechanisms, complicating clinical management and underscoring the urgent need for new therapeutic countermeasures.
A unique virulence mechanism of these pathogens is their ability to parasitize mammalian cells by intracellular replication and intercellular spread via membrane fusion, mediated by key secretion systems including the Bsa type III secretion system and the type VI secretion system T6SS-5. This intercellular lifecycle is critical for pathogenesis, and its disruption represents a novel therapeutic target.
To address this, the invention developed a high-throughput cell-based phenotypic screening assay targeting the entire Burkholderia intercellular lifecycle, including cell-cell fusion, using Burkholderia thailandensis (Bt) as a biosafety level 2 surrogate. Screening approximately 220,000 small molecules identified 268 hits that inhibit intercellular spread, with 32 validated to disrupt spread by Bp and/or Bm. Among these, burkfloxacin (BFX), a novel fluoroquinolone analog, inhibits intracellular bacterial replication, while flucytosine (5-FC), an FDA-approved antifungal, inhibits T6SS-5 secretion activity crucial for membrane fusion and intercellular spread.
Treatment with these compounds in a murine model of respiratory melioidosis significantly improved survival and decreased bacterial loads compared to the current standard of care, ceftazidime. Thus, the invention provides novel small molecule inhibitors that interfere with Burkholderia intercellular spread by targeting distinct aspects of their intracellular lifecycle, presenting promising therapeutic strategies against these Tier 1 select agent pathogens.
Claims Coverage
The patent contains one independent claim focused on a method of inhibiting Burkholderia intercellular spread using burkfloxacin, with several dependent claims expanding on formulations, dosing, and comparative efficacy.
Method of inhibiting intercellular spreading by Burkfloxacin
A therapeutic method comprising contacting Burkholderia pseudomallei or Burkholderia mallei bacteria with burkfloxacin at amounts sufficient to inhibit intercellular spread, including administration to patients diagnosed with melioidosis or glanders.
Formulation with pharmaceutically acceptable carriers
Incorporation of burkfloxacin in compositions further comprising pharmaceutically acceptable carriers such as pH adjusting agents, buffering agents, tonicity adjusting agents, wetting agents, antioxidants, viscosity-increasing agents, or preservatives.
In vivo administration to infected patients
Administration of the burkfloxacin-containing formulation to patients with Burkholderia infection, specifically dosing within specified ranges and durations to inhibit bacterial intercellular spread.
Dosing regimens for therapeutic efficacy
Providing treatment regimens administering burkfloxacin at doses between 1 mg/kg/day and 250 mg/kg/day, administered at least once daily for multiple days (4 to 7 or more).
Concentration range sufficient for inhibition
Utilization of burkfloxacin concentrations of at least 1 μM, 5 μM, or 10 μM sufficient to inhibit the intercellular spreading of Burkholderia species.
Superior efficacy compared to ceftazidime
Use of burkfloxacin showing greater inhibition of intercellular spread of Burkholderia compared to ceftazidime at concentrations from 0.125 μM to 8 μM.
The claims cover a method of suppressing Burkholderia intercellular spread by treatment with burkfloxacin including pharmaceutical compositions, dosing regimens, and superiority over existing therapies, providing a comprehensive therapeutic approach.
Stated Advantages
Compounds identified, including burkfloxacin and flucytosine, are significantly more effective than ceftazidime, the current drug of choice, at improving survival and reducing bacterial load in murine models of respiratory melioidosis.
The screening approach allows identification of small molecule inhibitors that are capable of penetrating both host and bacterial membranes, enabling inhibition of intracellular pathogens.
Burkfloxacin accumulates within host cells resulting in potent inhibition of intracellular Burkholderia growth at lower concentrations than ciprofloxacin.
Flucytosine inhibits the secretion activity of the type VI secretion system T6SS-5, targeting a unique virulence mechanism without significantly affecting intracellular bacterial replication or growth in vitro, minimizing selection pressure for resistance.
Documented Applications
Use of burkfloxacin and flucytosine for treatment of melioidosis and glanders caused by Burkholderia pseudomallei and Burkholderia mallei infections.
High-throughput cell-based phenotypic screening methods to identify small molecules that disrupt intracellular replication and intercellular spread of Burkholderia species.
Therapeutic regimens involving systemic administration of identified agents to patients diagnosed with melioidosis or glanders.
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