Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.

Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria.

Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely resistant to known antibiotics. AMR is caused by several mechanisms, among which the (over)expression of multidrug efflux pumps plays a central role. Multidrug efflux pumps are transmembrane transporters, naturally expressed by Gram-negative bacteria, able to extrude and confer resistance to several classes of antibiotics. Targeting them would be an effective way to revive various options for treatment. Many efflux pump inhibitors (EPIs) have been described in the literature; however, none of them have entered clinical trials to date. This review presents eight families of EPIs active against Escherichia coli or Pseudomonas aeruginosa. Structure-activity relationships, chemical synthesis, in vitro and in vivo activities, and pharmacological properties are reported. Their binding sites and their mechanisms of action are also analyzed comparatively.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

AcrB: a mean, keen, drug efflux machine.

Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.

Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump.

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

Bone-marrow-derived mesenchymal stem cells attenuate cognitive deficits in an endothelin-1 rat model of stroke.

PURPOSE: Stroke is the third leading cause of death and permanent disability in the United States, often producing long-term cognitive impairments, which are not easily recapitulated in animal models. The goals of this study were to assess whether: (1) the endothelin-1 (ET-1) model of chronic stroke produced discernable cognitive deficits; (2) a spatial operant reversal task (SORT) would accurately measure memory deficits in this model; and (3) bone-marrow-derived mesenchymal stem cells (BMMSCs) could reduce any observed deficits. METHODS: Rats were given unilateral intracerebral injections of vehicle or ET-1, a stroke-inducing agent, near the middle cerebral artery. Seven days later, they were given intrastriatal injections of BMMSCs or vehicle, near the ischemic penumbra. The cognitive abilities of the rats were assessed on a novel SORT, which was designed to efficiently distinguish cognitive deficits from potential motoric confounds. RESULTS: Rats given ET-1 had significantly more cognitive errors at six weeks post-stroke on the SORT, and that these deficits were attenuated by BMMSC transplants. CONCLUSIONS: These findings indicate that: (1) the ET-1 model produces chronic cognitive deficits; (2) the SORT efficiently measures cognitive deficits that are not confounded by motoric impairment; and (3) BMMSCs may be a viable treatment for stroke-induced cognitive dysfunction.

Switch-loop flexibility affects transport of large drugs by the promiscuous AcrB multidrug efflux transporter.

Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å(2) are less well transported than other substrates.

Chronic traumatic diaphragmatic hernia with pericardial rupture and associated gastroesophageal reflux.

Major thoracic and abdominal trauma damages the diaphragm 5% of the time. These injuries may be recognized when they occur but often are discovered months later during work up for related symptoms. Typically, the injury is to the left posterolateral aspect of the diaphragm. Rarely, rupture through the central diaphragmatic tendon into the pericardial space occurs and this results in different symptoms than the more common injury. We present the case of a patient who presented with chest pain, near syncopal episodes and refractory gastroesophageal reflux years after he was struck by a car and hospitalized. Radiographic imaging included a chest CT that demonstrated herniation of the transverse colon into the mediastinum. During exploration, a defect in the central diaphragm was found with free communication between the peritoneal and pericardial spaces. In this paper, we review our management of this unusual diaphragmatic hernia and the unique symptoms associated with it.

Upgrading the service mix in primary care practices may increase revenue.

Hospitals and health systems that own primary care physician networks want to optimize revenues when possible while still providing high-quality patient care. One way to accomplish this objective is to enhance the service mix for patients seen by the primary care practices. An analysis of the diagnosis and service mix of three primary care networks showed that the primary care practices mainly are involved with common acute and chronic conditions, which are associated with office visits that are paid at low- and mid-level rates. A comparison of the primary care networks' diagnosis and service mix with Medicare data on the mix of office visits for family practitioners, internists, and cardiologists showed, for example, that primary care physicians have more level 2 office visits and fewer level 4 office visits than cardiologists. By upgrading the patient office visits from the routine acute and chronic conditions associated with lower-level visits to more serious conditions associated with higher-level visits, primary care practices may increase total practice revenue.

Demographic and intrapartum characteristics of term pregnancies with early-onset neonatal seizures.

OBJECTIVE: To compare the demographic and intrapartum factors of term pregnancies in which early-onset neonatal seizures developed with the characteristics of a large, unselected control population. STUDY DESIGN: Pregnancies delivered at term (gestational age > or = 37 weeks) in one birthing unit between 1984 and 1995 with a discharge diagnosis of neonatal seizures were identified. Maternal and neonatal charts of these patients were reviewed to confirm the diagnosis of early-onset seizure (EOS) which was defined as a clinical or EEG-diagnosed seizure within 72 hours of life. Demographic and intrapartum factors were compared between these EOS cases and all singleton term pregnancies delivered over the same time period in which there was no EOS. A regression model was then developed to determine factors predictive of EOS. RESULTS: Of 80,561 total deliveries during the 11-year study period, there were 64,340 control and 62 EOS (0.1%) deliveries. Regression modeling identified NICU admission, depressed 1- and 5-minute Apgar scores, and neonatal intubation as predictors of EOS, but not operative vaginal, vaginal breech, or cesarean delivery. CONCLUSION: Depressed condition at birth and/or the requirement for NICU care was the most important risk associated with early seizures in term infants.

Regulation of expression and phosphorylation of A9/alpha 6 beta 4 integrin in normal and neoplastic keratinocytes.

The A9 antigen is a basement membrane antigen of normal squamous epithelial cells that is strongly expressed in many squamous carcinomas. High expression of this antigen is associated with early relapse in squamous cell carcinomas of the head and neck. We now know that the A9 antigen is structurally, immunologically, and functionally similar to the alpha 6 beta 4 integrin that has been shown to be linked to metastatic behavior in murine tumor models. The alpha 6 and beta 4 genes have been cloned and sequenced, and a model has been constructed from the deduced amino acid composition. In this study we present a hypothetical model and use it to design experiments to assess the factors that influence the expression of the A9/alpha 6 beta 4 integrin in normal and malignant keratinocytes. High calcium induces down regulation of A9/alpha 6 beta 4 antigen in normal but not malignant keratinocytes within 24 hours. Although calcium can down-regulate beta 4 message in tumor cells in the absence of epidermal growth factor (EGF), transcription of beta 4 increased in the tumor cells under the conditions we used for assessing antigen expression (calcium plus EGF). Retinoic acid also stimulated transcription of beta 4 in tumor cells, but this was partially inhibited by the presence of high calcium. Phosphorylation of the beta 4 chain was stimulated by epidermal growth factor and calcium in normal keratinocytes, but in the malignant cells phosphorylation was constant regardless of the culture conditions. Our results indicate that high expression of the alpha 6 beta 4 integrin is associated with conditions that favor migration and undifferentiated proliferation of normal keratinocytes and that malignant keratinocytes differ from normal keratinocytes by constitutive phosphorylation of beta 4 and by failure to downregulate beta 4 transcription in response to calcium in the presence of EGF.

Looking beyond cost containment.

Legislators, third parties, physicians, and patients alike have spent countless hours in recent years searching for a way to contain rising medical costs. Here we look at how controlling overutilization of services may lead to cost containment.