Development of Anti-Infectives Targeting Bacterial LpxH or Fungal FKBP12-Calcineurin

Abstract

Anti-infectives are critical tools for the treatment of infectious diseases, but the use of anti-infectives drives resistance. These resistant infections pose a serious public health threat due to the difficulty in treating them. In spite of this, few new anti-infectives are entering into therapeutic use, meaning that anti-infective resistance is outpacing development. The development of new anti-infectives is necessary to address this rising anti-infective resistance. Of the anti-infective classes, antibiotics and antifungals are of special interest, owing to the broad emergence of antibiotic resistance and the limited tools available for antifungal treatments. To address the issue of antifungal resistance, we have advanced the development of a new class of antifungals target calcineurin, a phosphatase enzyme responsible for the fungal stress response and survival in human infections. Though the inhibition of calcineurin is potently fungicidal, calcineurin inhibition has been precluded as an antifungal target due to conservation of calcineurin in humans. Human calcineurin is responsible for activation of the immune system; this activity is incompatible with antifungal targeting. There are, however, structural differences between human and fungal calcineurin that may be exploited to design calcineurin inhibitors that maintain antifungal activity while abolishing deleterious immunosuppressive activity. Modification of FK506 and FK520, natural products that inhibit both human and fungal calcineurin, may enable selective activity for only fungal inhibition of calcineurin. This work details the synthesis, characterization, and biological evaluation of a series of C32, C22, and C18 modified derivatives of FK520 with antifungal activity and reduced immunosuppressive activity. This work has identified both C22 and C32 modified compounds that maintain antifungal activity against Candida albicans and Cryptococcus neoformans in vitro while reducing iv immunosuppressive activity. In doing so, this work advances the development of calcineurin as a target for antifungal development. Gram-negative bacterial infections are especially difficult to treat owing to their outer membrane, which limits the permeation of antibiotics into cell. The biosynthesis of components of this outer membrane are not conserved in humans and may be targeted for antibiotic developments. The anchor component of the lipopolysaccharide layer of the outer membrane is produced by the Raetz pathway; the phosphatase enzyme of the Raetz pathway is of particular interest for antibiotic development as its inhibition both disrupts the bacterial outer membrane and leads to the accumulation of toxic glucosamine substrates in the bacterial cell, a dual mechanism of bactericidal activity. Previous work has identified a novel LpxH inhibitor AZ1. This work details the development, optimization, characterization, and biological evaluation of a series of LpxH inhibitors, focusing on improvements along four axes: metal chelation of LpxH inhibitors to LpxH’s dimanganese cluster active site, modification of the trifluoromethylphenyl group to improve activity and spectrum of activity, targeting binding to critical LpxH residues ARG80 and ASN79, and varied modifications to improve membrane permeability. These avenues of research have led to the development of LpxH inhibitors with potent in vitro activity against Klebsiella pneumoniae and Escherichia coli and sub-nanomolar inhibition of the LpxH enzyme. In doing so, this work expands the utility of LpxH inhibition as an antibiotic target.