A host-directed macrocyclic peptide therapeutic for MDR gram negative bacterial infections.

The emergence of infections by carbapenem resistant Enterobacteriaceae (CRE) pathogens has created an urgent public health threat, as carbapenems are among the drugs of last resort for infections caused by a growing fraction of multi-drug resistant (MDR) bacteria. There is global consensus that new preventive and therapeutic strategies are urgently needed to combat the growing problem of MDR bacterial infections. Here, we report on the efficacy of a novel macrocyclic peptide, minimized theta-defensin (MTD)-12813 in CRE sepsis. MTD12813 is a theta-defensin inspired cyclic peptide that is highly effective against CRE pathogens K. pneumoniae and E. coli in vivo. In mouse septicemia models, single dose administration of MTD12813 significantly enhanced survival by promoting rapid host-mediated bacterial clearance and by modulating pathologic cytokine responses, restoring immune homeostasis, and preventing lethal septic shock. The peptide lacks direct antibacterial activity in the presence of mouse serum or in peritoneal fluid, further evidence for its indirect antibacterial mode of action. MTD12813 is highly stable in biological matrices, resistant to bacterial proteases, and nontoxic to mice at dose levels 100 times the therapeutic dose level, properties which support further development of the peptide as a first in class anti-infective therapeutic.

Rhesus Theta Defensin 1 Promotes Long Term Survival in Systemic Candidiasis by Host Directed Mechanisms.

Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections in hospitalized and immunosuppressed patients. Mortality rates associated with these infections have risen sharply due to the emergence of multidrug resistant (MDR) strains of C. albicans and other Candida spp., highlighting the urgent need of new antifungal therapies. Rhesus theta (θ) defensin-1 (RTD-1), a natural macrocyclic antimicrobial peptide, was recently shown to be rapidly fungicidal against clinical isolates of MDR C. albicans in vitro. Here we found that RTD-1 was rapidly fungicidal against blastospores of fluconazole/caspofungin resistant C. albicans strains, and was active against established C. albicans biofilms in vitro. In vivo, systemic administration of RTD-1, initiated at the time of infection or 24 h post-infection, promoted long term survival in candidemic mice whether infected with drug-sensitive or MDR strains of C. albicans. RTD-1 induced an early (4 h post treatment) increase in neutrophils in naive and infected mice. In vivo efficacy was associated with fungal clearance, restoration of dysregulated inflammatory cytokines including TNF-α, IL-1ß, IL-6, IL-10, and IL-17, and homeostatic reduction in numbers of circulating neutrophils and monocytes. Because these effects occurred using peptide doses that produced maximal plasma concentrations (Cmax) of less than 1% of RTD-1 levels required for in vitro antifungal activity in 50% mouse serum, while inducing a transient neutrophilia, we suggest that RTD-1 mediates its antifungal effects in vivo by host directed mechanisms rather than direct fungicidal activity. Results of this study suggest that θ-defensins represent a new class of host-directed compounds for treatment of disseminated candidiasis.

Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion.

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.