The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.

Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.

Abstract. We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca 2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (or) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and coiocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.ROTEIN kinase C (PKC)', the Ca 2÷ and phospholipiddependent kinase is recognized to be a key regulatory enzyme. Several growth factors and hormones which are essential for normal cell growth and differentiated function regulate the activation state of PKC (26). This is due to their ability to stimulate phospholipase C activity, and consequently, to increase cellular diacylglycerol, an endogenous regulator of PKC. Tumor-promoting phorbol esters mimic diacylglycerol; they also bind and activate PKC (7, 25). Therefore, prolonged activation of PKC by phorbol esters, which is used in tumor promotion protocols, is thought to play a major role in tumorigenesis. These data demonstrate the importance of PKC both in normal cell function and in carcinogenesis.These results, which underscore the key regulatory role of PKC, have led to efforts to identify substrates for PKC which are relevant to the observed biological responses. Identification of specific substrates has been complicated both by the demonstration that many proteins can serve as PKC substrates in vitro and by the lack of specific PKC inhibitors. Furthermore, because phorbol esters and PKC activation have been linked to a wide variety of biological responses, it has been difficult to distinguish between primary and secondary effects of PKC action (26).PKC is now recognized to be a group of enzymes encoded by a family of genes (reviewed in reference 27). If the isozymes have different substrate specificities, then PKC heterogeneity may also complicate identification of PKC substrates. Although little data exist to directly compare isozyme 1. Abbreviations used in this paper: CSK, cytoskeleton; PDBu, phorbol dibutyrate; PKC, protein kinase C. substrate specificities, evidence for differential ability to phosphorylate the EGF receptor has been presented (14). Subtle differences in cofactor requirements among the isozymes have also been reported (16, 31). H...

Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of[3H]phorbol 12,13-dibutyrate ([3H]PBt2) in a dose-dependent fashion to <10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6 ± 0.8 aug/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the K1 determined for diolein was independent of the diolein concentrations over the range of 1.5-80 pug/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The K; of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 ,ug/ml, for example, the K; was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (it) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.The phorbol esters are the most intensively studied class of highly potent mouse skin tumor promoters (1). In addition to their activity as tumor promoters, the phorbol esters have profound biological effects in many systems (2-5). They cause partial mimicry of the transformed phenotype in normal cells; they synergize with growth factors; and they affect cellular differentiation, inhibiting differentiation in some systems and inducing it in others. This laboratory (6) and others to demonstrate and characterize phorbol ester receptors in tissue preparations and intact cells. Structure-activity analysis strongly argues that these receptors mediate biological responses to the phorbol esters (9).The tissue distribution (10, 11), evolutionary conservation (10), and phospholipid requirements (12) for the phorbol ester receptor markedly resembled those of the Ca2+-phospholipid dependent. protein kinase (protein kinase C) described by Nishizuka and co-workers (13) and by Kuo and co-workers (14, 15). Nishizuka's group, together with M. Castagna, have examined the effect of the phorbol esters directly on this kinase (16); they observed that the phorbol esters caused activation of kinase activity, apparently by shifting th...

Altered subcellular distribution and activity of protein kinase C (PKC) is associated with transmembrane signalling in a variety of systems in which receptor occupancy leads to increased hydrolysis of polyphosphoinositides. Here we report evidence that in B lymphocytes, cyclic-cAMP-generating signal transduction pathways can activate translocation of PKC from the cytosol to the nucleus. Elevated cAMP levels and translocation of PKC to the nucleus are induced by antibodies against Ia antigens in normal B lymphocytes. Further, cAMP analogues mediate the translocation of PKC to the nucleus of these cells. These findings suggest that in physiological situations, ligation of B-lymphocyte Ia molecules by helper T cells leads to increased cAMP production which in turn causes PKC translocation to the nucleus. In view of recent observations that antibodies against Ia antigens induce differentiation of B cells, we conclude that nuclear PKC may function in the regulation of gene expression.