Triple combinations of carbapenem, penicillin and β-lactamase inhibitor antibiotic classes are synergistic against MRSA through a mechanism involving allostery-based synergy and collateral sensitivity and can thus be applied at doses that lead to less resistance. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple β-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other clinical MRSA isolates in vitro and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem-penicillin–β-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to β-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older β-lactam antibiotics could be effective against MRSA infections in humans.

Gliomas are differentiated into two major disease subtypes, astrocytoma or oligodendroglioma, which are then characterized as either IDH (isocitrate dehydrogenase)-wild type or IDH-mutant due to the dramatic differences in prognosis and overall survival. Here, we investigated the genetic background of IDH1-mutant gliomas using the Catalogue of Somatic Mutations in Cancer (COSMIC) database. In astrocytoma patients, we found that IDH1 is often co-mutated with TP53, ATRX, AMBRA1, PREX1, and NOTCH1, but not CHEK2, EGFR, PTEN, or the zinc finger transcription factor ZNF429. The majority of the mutations observed in these genes were further confirmed to be either drivers or pathogenic by the Cancer-Related Analysis of Variants Toolkit (CRAVAT). Gene expression analysis showed down-regulation of DRG2 and MSN expression, both of which promote cell proliferation and invasion. There was also significant over-expression of genes such as NDRG3 and KCNB1 in IDH1-mutant astrocytoma patients. We conclude that IDH1-mutant glioma is characterized by significant genetic changes that could contribute to a better prognosis in glioma patients.

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the μ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT G protein–coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily–selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the μ-opioid receptor most efficaciously because it has the ability to cross cell membranes.

Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1'-(2'-oleoyl-3'-hydroxy)-glycerol (S,S-(2,2',C_18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3',C_18:1)-BMP>R,S-(3,1',C_18:1)-BMP>R,R-(1,1',C_18:1)>S,S-(2,2')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC₅₀s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 μM and approximately 30 μM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC₅₀s ranging from 6.8 to 63.3 μM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.