Mapping N- to C-terminal allosteric coupling through disruption of a putative CD74 activation site in D-dopachrome tautomerase.

The macrophage migration inhibitory factor (MIF) protein family consists of MIF and D-dopachrome tautomerase (also known as MIF-2). These homologs share 34% sequence identity while maintaining nearly indistinguishable tertiary and quaternary structure, which is likely a major contributor to their overlapping functions, including the binding and activation of the cluster of differentiation 74 (CD74) receptor to mediate inflammation. Previously, we investigated a novel allosteric site, Tyr99, that modulated N-terminal catalytic activity in MIF through a "pathway" of dynamically coupled residues. In a comparative study, we revealed an analogous allosteric pathway in MIF-2 despite its unique primary sequence. Disruptions of the MIF and MIF-2 N termini also diminished CD74 activation at the C terminus, though the receptor activation site is not fully defined in MIF-2. In this study, we use site-directed mutagenesis, NMR spectroscopy, molecular simulations, in vitro and in vivo biochemistry to explore the putative CD74 activation region of MIF-2 based on homology to MIF. We also confirm its reciprocal structural coupling to the MIF-2 allosteric site and N-terminal enzymatic site. Thus, we provide further insight into the CD74 activation site of MIF-2 and its allosteric coupling for immunoregulation.

α1,3-Fucosyltransferase-IX, an enzyme of pulmonary endogenous lung stem cell marker SSEA-1, alleviates experimental bronchopulmonary dysplasia.

BACKGROUND: Endogenous pulmonary stem cells (PSCs) play an important role in lung development and repair; however, little is known about their role in bronchopulmonary dysplasia (BPD). We hypothesize that an endogenous PSC marker stage-specific embryonic antigen-1 (SSEA-1) and its enzyme, α1,3-fucosyltransferase IX (FUT9) play an important role in decreasing inflammation and restoring lung structure in experimental BPD. METHODS: We studied the expression of SSEA-1, and its enzyme FUT9, in wild-type (WT) C57BL/6 mice, in room air and hyperoxia. Effects of intraperitoneal administration of recombinant human FUT9 (rhFUT9) on lung airway and parenchymal inflammation, alveolarization, and apoptosis were evaluated. RESULTS: On hyperoxia exposure, SSEA-1 significantly decreased at postnatal day 14 in hyperoxia-exposed BPD mice, accompanied by a decrease in FUT9. BPD and respiratory distress syndrome (RDS) in human lungs showed decreased expression of SSEA-1 as compared to their term controls. Importantly, intraperitoneal administration of FUT9 in the neonatal BPD mouse model resulted in significant decrease in pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leakage, alveolar simplification, and cell death in the hyperoxia-exposed BPD mice. CONCLUSIONS: An important role of endogenous PSC marker SSEA-1 and its enzyme FUT9 is demonstrated, indicating early systemic intervention with FUT9 as a potential therapeutic option for BPD. IMPACT: Administration of rhFUT9, an enzyme of endogenous stem cell marker SSEA-1, reduces pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leak and cell death in the BPD mouse model. SSEA-1 is reported for the first time in experimental BPD models, and in human RDS and BPD. rhFUT9 treatment ameliorates hyperoxia-induced lung injury in a developmentally appropriate BPD mouse model. Our results have translational potential as a therapeutic modality for BPD in the developing lung.

Chitin-Derived AVR-48 Prevents Experimental Bronchopulmonary Dysplasia (BPD) and BPD-Associated Pulmonary Hypertension in Newborn Mice.

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and a key contributor to the large health care burden associated with prematurity, longer hospital stays, higher hospital costs, and frequent re-hospitalizations of affected patients through the first year of life and increased resource utilization throughout childhood. This disease is associated with abnormal pulmonary function that may lead to BPD-associated pulmonary hypertension (PH), a major contributor to neonatal mortality and morbidity. In the absence of any definitive treatment options, this life-threatening disease is associated with high resource utilization during and after neonatal intensive care unit (NICU) stay. The goal of this study was to test the safety and efficacy of a small molecule derivative of chitin, AVR-48, as prophylactic therapy for preventing experimental BPD in a mouse model. Two doses of AVR-48 were delivered either intranasally (0.11 mg/kg), intraperitoneally (10 mg/kg), or intravenously (IV) (10 mg/kg) to newborn mouse pups on postnatal day (P)2 and P4. The outcomes were assessed by measuring total inflammatory cells in the broncho-alveolar lavage fluid (BALF), chord length, septal thickness, and radial alveolar counts of the alveoli, Fulton's Index (for PH), cell proliferation and cell death by immunostaining, and markers of inflammation by Western blotting and ELISA. The bioavailability and safety of the drug were assessed by pharmacokinetic and toxicity studies in both neonatal mice and rat pups (P3-P5). Following AVR-48 treatment, alveolar simplification was improved, as evident from chord length, septal thickness, and radial alveolar counts; total inflammatory cells were decreased in the BALF; Fulton's Index was decreased and lung inflammation and cell death were decreased, while angiogenesis and cell proliferation were increased. AVR-48 was found to be safe and the no-observed-adverse-effect level (NOAEL) in rat pups was determined to be 100 mg/kg when delivered via IV dosing with a 20-fold safety margin. With no reported toxicity and with a shorter half-life, AVR-48 is able to reverse the worsening cardiopulmonary phenotype of experimental BPD and BPD-PH, compared to controls, thus positioning it as a future drug candidate.

Hyperoxia-Induced miR-195 Causes Bronchopulmonary Dysplasia in Neonatal Mice.

Background: Exposure to hyperoxia is an important factor in the development of bronchopulmonary dysplasia (BPD) in preterm newborns. MicroRNAs (miRs) have been implicated in the pathogenesis of BPD and provide a potential therapeutic target. Methods: This study was conducted utilizing a postnatal animal model of experimental hyperoxia-induced murine BPD to investigate the expression and function of miR-195 as well as its molecular signaling targets within developing mouse lung tissue. Results: miR-195 expression levels increased in response to hyperoxia in male and female lungs, with the most significant elevation occurring in 40% O2 (mild) and 60% O2 (moderate) BPD. The inhibition of miR-195 improved pulmonary morphology in the hyperoxia-induced BPD model in male and female mice with females showing more resistance to injury and better recovery of alveolar chord length, septal thickness, and radial alveolar count. Additionally, we reveal miR-195-dependent signaling pathways involved in BPD and identify PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) as a novel specific target protein of miR-195. Conclusions: Our data demonstrate that high levels of miR-195 in neonatal lungs cause the exacerbation of hyperoxia-induced experimental BPD while its inhibition results in amelioration. This finding suggests a therapeutic potential of miR-195 inhibition in preventing BPD.

2,5-Pyridinedicarboxylic acid is a bioactive and highly selective inhibitor of D-dopachrome tautomerase.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are two pleotropic cytokines, which are coexpressed in various cell types to activate the cell surface receptor CD74. Via the MIF/CD74 and D-DT/CD74 axes, the two proteins exhibit either beneficial or deleterious effect on human diseases. In this study, we report the identification of 2,5-pyridinedicarboxylic acid (a.k.a. 1) that effectively blocks the D-DT-induced activation of CD74 and demonstrates an impressive 79-fold selectivity for D-DT over MIF. Crystallographic characterization of D-DT-1 elucidates the binding features of 1 and reveals previously unrecognized differences between the MIF and D-DT active sites that explain the ligand's functional selectivity. The commercial availability, low cost, and high selectivity make 1 the ideal tool for studying the pathophysiological functionality of D-DT in disease models. At the same time, our comprehensive biochemical, computational, and crystallographic analyses serve as a guide for generating highly potent and selective D-DT inhibitors.

Chitin Analog AVR-25 Prevents Experimental Bronchopulmonary Dysplasia.

Infants born extremely preterm are at a high risk of developing bronchopulmonary dysplasia (BPD) which is characterized by large, simplified alveoli, increased inflammation, disrupted and dysregulated vasculogenesis, decreased cell proliferation, and increased cell death in the lungs. Due to lack of specific drug treatments to combat this condition, BPD and its long-term complications have taken a significant toll of healthcare resources. AVR-25, a novel immune modulator experimental compound, was able to partially recover the pulmonary phenotype in the hyperoxia-induced experimental mouse model of BPD. We anticipate that AVR-25 will have therapeutic potential for managing human BPD.

Small Molecule Inhibitor Adjuvant Surfactant Therapy Attenuates Ventilator- and Hyperoxia-Induced Lung Injury in Preterm Rabbits.

BACKGROUND: Invasive mechanical ventilation (IMV) has become one of the mainstays of therapy in NICUs worldwide, as a result of which premature babies with extremely low birth weight have been able to survive. Although lifesaving, IMV can result in lung inflammation and injury. Surfactant therapy is considered a standard of care in preterm infants with immature lungs. Recently, small molecule inhibitors like siRNAs and miRNAs have been used for therapeutic purposes. Ddit3 (CHOP), Ang2 and miR34a are known to be upregulated in experimental lung injury. We wanted to test whether inhibitors for these molecules (CHOP siRNA, Ang2 siRNA, and miR34a antagomir) if used alone or with a combination with surfactant (Curosurf®) would help in reducing ventilation and hyperoxia-induced injury in an experimental lung injury model. METHODS: Preterm rabbits born by cesarean section were intratracheally instilled with the three small molecule inhibitors with or without Curosurf® prior to IMV and hyperoxia exposure. Prior to testing the inhibitors in rabbits, these small molecule inhibitors were transfected in mouse lung epithelial cells (MLE12 and AECII) and delivered to neonatal mouse pups intranasally as a proof of concept that surfactant (Curosurf®) could be used as an effective vehicle for administration of such drugs. Survival, pulmonary function tests, histopathology, immunostaining, quantitative PCR and western blotting were done to see the adjuvant effect of surfactant with these three small molecule inhibitors. RESULTS: Our data shows that Curosurf® can facilitate transfection of small molecules in MLE12 cells with the same and/or increased efficiency as Lipofectamine. Surfactant given alone or as an adjuvant with small molecule inhibitors increases survival, decreases IMV and hyperoxia-induced inflammation, improves pulmonary function and lung injury scores in preterm rabbit kits. CONCLUSION: Our study shows that Curosurf® can be used successfully as an adjuvant therapy with small molecule inhibitors for CHOP/Ang2/miR34a. In this study, of the three inhibitors used, miR34a inhibitor seemed to be the most promising compound to combat IMV and hyperoxia-induced lung injury in preterm rabbits.