A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism

Abstract

Aspergillus fumigatus can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an A. fumigatus wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, and two were polyaminocarboxylates. All three groups showed good in vitro activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with in vivo fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy.

Keywords: Aspergillus fumigatus; animal models; antifungal agents; antifungal susceptibility testing; aspergillosis; bioluminescence; drug screening; fungal infection; immunosuppression; zinc metabolism.

FIG 1

Percent inhibition based on luminescence measurements of A. fumigatus wild type (AF14) grown either with no added ions or with the addition of 100 μM CuSO₄, FeSO₄, MgSO₄, MnSO₄, or ZnSO₄ for 15 h in the presence of the polyaminocarboxylate Ami03 (A) or Ami04 (B).

FIG 2

Hyphal length percent inhibition of A. fumigatus wild type (AF14) or triple zinc transporter knockout (AF721) grown either with no added zinc or with 100 μM ZnSO₄ for 10 h in the presence of the polyaminocarboxylate Ami03 (A) or Ami04 (B).

FIG 3

Percent inhibition based on luminescence measurements of A. fumigatus wild type (AF14) grown in the presence of the polyaminocarboxylate Ami03 (A) or Ami04 (B). Removed at 8 h indicates that medium was replaced with fresh medium containing no tested compound after 8 h of incubation. Added at 8 h indicates that compounds were added to the medium after 8 h of incubation. The cultures were incubated for an additional 7 h, resulting in a total incubation time of 15 h.

FIG 4

Percent survival (A) and luminescence (in photons per second) (B) of immunosuppressed mice that were intranasally infected with 7.5 × 10⁴ A. fumigatus wild-type (AF14) conidia and treated with the polyaminocarboxylate Ami04. A dose of 15 mg/kg/day was able to significantly improve mouse survival (P = 0.0024) and resulted in a 46% reduction in luminescence on day 3 and 52% reduction on day 5 compared to the control group.

FIG 5

Examples showing luminescence of mice treated with 7.5 or 15 mg/kg/day of the polyaminocarboxylate Ami04 and of a DMSO placebo group. Mice in all three groups developed aspergillosis; however, only the ones receiving 15 mg/kg/day showed 100% survival. Min, minimum; Max, maximum.

FIG 6

Treated mice displayed less severe lung invasion by the fungus. Control mice displayed very heterogeneous lesions from small necrotic foci (black arrowhead) (A), with destruction of bronchiole epithelium (black arrowhead) (B and C), and blood vessel invasion by the fungus (D and E), to randomly distributed multifocal abscesses containing hyphae (F). Most treated mice (6/10) displayed minimal to mild inflammatory lesions, characterized by perivascular lymphocyte and plasma cell infiltrates (black arrowheads) (G and H), with no fungi invading the parenchyma (I). Less frequently, mice (4/10) displayed randomly distributed inflammatory (with neutrophils) (J and K) or necrotic lesions (black arrowhead), with few intralesional fungi (black arrowhead) (L). Panels A, B, D, E, G, H, J, and K represent hematoxylin & eosin (HE) staining, and panels C, F, I, and L represent Grocott-Gomori's staining.