Long Term Chronic <em>Pseudomonas aeruginosa</em> Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

San Raffaele Scientific Institute

Italian Cystic Fibrosis Research Foundation

A series of novel S-DABO analogues, characterized by different substitution patterns at positions 2, 5, and 6 of the heterocyclic ring, were synthesized in a straightforward fashion by means of parallel synthesis and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Most of the compounds proved to be highly active on the wild-type enzyme both in enzymatic and cellular assays, with one of them emerging as the most active reverse transcriptase inhibitor reported so far (EC50wt=25 pM). The general loss of potency displayed by the compounds toward clinically relevant mutant strains was deeply studied through a molecular modeling approach, leading to the evidence that the dynamic of the entrance in the non-nucleoside binding pocket could represent the basis of the inhibitory activity of the molecules.

Dihydro-alkylthio-benzyl-oxopyrimidines as inhibitors of reverse transcriptase: synthesis and rationalization of the biological data on both wild-type enzyme and relevant clinical mutants.

We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis. A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation. These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling.

We investigated some pyrrolobenzoxazepinone (PBOs, 3e-i) analogues of early described effective non-nucleoside inhibitors of HIV-1 reverse transcriptase (RT). Enzymological studies of 3e-i enantiomers, with wild type (wt) RT and some drug-resistant mutants, revealed a stereoselective mode of action and selectivity for RT ternary complex. Unexpectedly (+)-3g was found more potent towards the L100I mutant than towards the wt RT, whereas (+)-3h inhibited the K103N mutant and RT wt with comparable potency.

Enantioselective binding of second generation pyrrolobenzoxazepinones to the catalytic ternary complex of HIV-1 RT wild-type and L100I and K103N drug resistant mutants.

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

Matrix disruptions, growth, and degradation of cartilage with impaired sulfation.

Although antibiotic resistance represents a public health emergency, the pipeline of new antibiotics is running dry. Repurposing of old drugs for new clinical applications is an attractive strategy for drug development. We used the bacterial pathogen Pseudomonas aeruginosa as a target for the screening of antivirulence activity among marketed drugs. We found that the antimycotic agent flucytosine inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major P. aeruginosa virulence factors, namely pyoverdine, PrpL protease, and exotoxin A. Flucytosine administration at clinically meaningful dosing regimens suppressed P. aeruginosa pathogenicity in a mouse model of lung infection. The in vitro and in vivo activity of flucytosine against P. aeruginosa, combined with its desirable pharmacological properties, paves the way for clinical trials on the anti-P. aeruginosa efficacy of flucytosine in humans.

Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity.

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis.

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

Marcella Facchini

Infections and Cystic Fibrosis Unit,
Division of Immunology,
Transplantation and Infectious Diseases,
Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases

San Raffaele Scientific Institute

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Marcella Facchini

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San Raffaele Scientific Institute

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Infections and Cystic Fibrosis Unit,
Division of Immunology,
Transplantation and Infectious Diseases,
Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases