MSDC-0602K, a metabolic modulator directed at the core pathology of non-alcoholic steatohepatitis.

INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is a serious form of non-alcoholic fatty liver disease (NAFLD) for which there is no marketed treatments. NAFLD is initiated by excess intake of nutrients and recent evidence has pinpointed the mitochondrial pyruvate carrier (MPC) as a mediator of the nutritional overload signals. Areas covered: An overview is given of MSDC-0602K, a new agent in development that modulates the MPC and as such treats the symptoms of fatty liver including dysfunctional lipid metabolism, inflammation, and insulin resistance as well as the key liver pathology including fibrosis. METHODOLOGY: The current evaluation is written from the direct experience of the authors and review of published literature using standard search techniques. Expert Opinion: The mechanism of action of MSDC-0602K appears to be suited for treatment of the NASH pathophysiology. An ongoing phase 2b dose-ranging trial should demonstrate whether or not MSDC-0602K has the potential to be a cornerstone metabolic therapy for the treatment of NASH.

The beneficial metabolic effects of insulin sensitizers are not attenuated by mitochondrial pyruvate carrier 2 hypomorphism.

NEW FINDINGS: What is the central question of this study? The antidiabetic effects of thiazolidinedione (TZD) drugs may be mediated in part by a molecular interaction with the constituent proteins of the mitochondrial pyruvate carrier complex (MPC1 and MPC2). We examined the ability of a mutant mouse strain expressing an N-terminal truncation of MPC2 (Mpc2Δ16 mice) to respond to TZD treatment. What is the main finding and its importance? The response of Mpc2Δ16 mice to TZD treatment was not significantly different from that of wild-type C57BL6/J control animals, suggesting that the 16 N-terminal amino acids of MPC2 are dispensable for the effects of TZD treatment. Rosiglitazone and pioglitazone are thiazolidinedione (TZD) compounds that have been used clinically as insulin-sensitizing drugs and are generally believed to mediate their effects via activation of the peroxisome proliferator-activated receptor Î³ (PPARγ). Recent work has shown that it is possible to synthesize TZD compounds with potent insulin-sensitizing effects and markedly diminished affinity for PPARγ. Both clinically used TZDs and investigational PPARγ-sparing TZDs, such as MSDC-0602, interact with the mitochondrial pyruvate carrier (MPC) and inhibit its activity. The MPC complex is composed of two proteins, MPC1 and MPC2. Herein, we used mice expressing a hypomorphic MPC2 protein missing 16 amino acids in the N-terminus (Mpc2Δ16 mice) to determine the effects of these residues in mediating the insulin-sensitizing effects of TZDs in diet-induced obese mice. We found that both pioglitazone and MSDC-0602 elicited their beneficial metabolic effects, including improvement in glucose tolerance, attenuation of hepatic steatosis, reduction of adipose tissue inflammation and stimulation of adipocyte browning, in both wild-type and Mpc2Δ16 mice after high-fat diet feeding. In addition, truncation of MPC2 failed to attenuate the interaction between TZDs and the MPC in a bioluminescence resonance energy transfer-based assay or to affect the suppression of pyruvate-stimulated respiration in cells. Collectively, these data suggest that the interaction between TZDs and MPC2 is not affected by loss of the N-terminal 16 amino acids nor are these residues required for the insulin-sensitizing effects of these compounds.

Treating fatty liver disease by modulating mitochondrial pyruvate metabolism.

Modifying the entry of pyruvate into mitochondria may provide a unique approach to treat metabolic disease. The pharmacology of a new class of insulin sensitizers directed against a newly identified mitochondrial target may treat many aspects of nonalcoholic steatohepatitis, including fibrosis. This commentary suggests treating nonalcoholic steatohepatitis through a newly identified mechanism consistent with pathophysiology. (Hepatology Communications 2017;1:193-197).

Targeting the mitochondrial pyruvate carrier attenuates fibrosis in a mouse model of nonalcoholic steatohepatitis.

Diseases of the liver related to metabolic syndrome have emerged as the most common and undertreated hepatic ailments. The cause of nonalcoholic fatty liver disease is the aberrant accumulation of lipid in hepatocytes, though the mechanisms whereby this leads to hepatocyte dysfunction, death, and hepatic fibrosis are still unclear. Insulin-sensitizing thiazolidinediones have shown efficacy in treating nonalcoholic steatohepatitis (NASH), but their widespread use is constrained by dose-limiting side effects thought to be due to activation of the peroxisome proliferator-activated receptor γ. We sought to determine whether a next-generation thiazolidinedione with markedly diminished ability to activate peroxisome proliferator-activated receptor γ (MSDC-0602) would retain its efficacy for treating NASH in a rodent model. We also determined whether some or all of these beneficial effects would be mediated through an inhibitory interaction with the mitochondrial pyruvate carrier 2 (MPC2), which was recently identified as a mitochondrial binding site for thiazolidinediones, including MSDC-0602. We found that MSDC-0602 prevented and reversed liver fibrosis and suppressed expression of markers of stellate cell activation in livers of mice fed a diet rich in trans-fatty acids, fructose, and cholesterol. Moreover, mice with liver-specific deletion of MPC2 were protected from development of NASH on this diet. Finally, MSDC-0602 directly reduced hepatic stellate cell activation in vitro, and MSDC-0602 treatment or hepatocyte MPC2 deletion also limited stellate cell activation indirectly by affecting secretion of exosomes from hepatocytes. CONCLUSION: Collectively, these data demonstrate the effectiveness of MSDC-0602 for attenuating NASH in a rodent model and suggest that targeting hepatic MPC2 may be an effective strategy for pharmacologic development. (Hepatology 2017;65:1543-1556).

Mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration in experimental models of Parkinson's disease.

Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of Parkinson's disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neurodegeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP+) insult in murine and cultured human midbrain dopamine neurons and in an α-synuclein-based Caenorhabditis elegans model. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, MSDC-0160 improved locomotor behavior, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels, and reduced neuroinflammation. Long-term targeting of MPC preserved motor function, rescued the nigrostriatal pathway, and reduced neuroinflammation in the slowly progressive Engrailed1 (En1+/-) genetic mouse model of PD. Targeting MPC in multiple models resulted in modulation of mitochondrial function and mTOR signaling, with normalization of autophagy and a reduction in glial cell activation. Our work demonstrates that changes in metabolic signaling resulting from targeting MPC were neuroprotective and anti-inflammatory in several PD models, suggesting that MPC may be a useful therapeutic target in PD.

Loss of Mitochondrial Pyruvate Carrier 2 in the Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling.

Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite for gluconeogenesis in hepatocytes, which is important for the maintenance of normoglycemia during prolonged food deprivation but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2(-/-)) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte conversion of labeled pyruvate to TCA cycle intermediates and glucose. Unbiased metabolomic analyses of livers from fasted LS-Mpc2(-/-) mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for the loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2(-/-) hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import.

Mitochondrial pyruvate carrier 2 hypomorphism in mice leads to defects in glucose-stimulated insulin secretion.

Carrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2) is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2(Δ16)) was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2(Δ16) mice. Additionally, compared with wild-type controls, Mpc2(Δ16) mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic ß cell glucose sensing.

Insulin sensitizers in 2013: new insights for the development of novel therapeutic agents to treat metabolic diseases.

Insulin-sensitizing thiazolidinediones (TZDs) correct a root cause of type 2 diabetes and potentially other diseases of metabolic dysfunction, including conditions ranging from oncologic, inflammatory, and neurodegenerative diseases. Importantly, compounds with this mode of action can modify disease progression, as opposed to simply mitigating symptoms. However, side effects have limited the use of marketed agents. Moreover, the same and additional issues have prevented development of newer agents, and no new compounds with this mode of action have been approved since 1999. Here we briefly review the drug discovery track record of compounds in the TZD class as well as several classes of compounds that have been designed with substitutes for the TZD ring, while maintaining and/or expanding the ability to directly activate peroxisome proliferator-activated receptor (PPAR) transcription factors. A key discovery that could change the course of drug discovery in this area is a newly identified mitochondrial target for the insulin sensitizers. This has allowed new drug discovery into molecules designed to maintain this mitochondrial interaction while specifically avoiding significant interactions with PPAR receptors. This commentary suggests that a fresh approach could pave the way for a new directed group of therapeutic agents with potential for disease modification of common metabolic disorders.

Novel insulin sensitizer modulates nutrient sensing pathways and maintains ß-cell phenotype in human islets.

Major bottlenecks in the expansion of human ß-cell mass are limited proliferation, loss of ß-cell phenotype, and increased apoptosis. In our previous studies, activation of Wnt and mTOR signaling significantly enhanced human ß-cell proliferation. However, isolated human islets displayed insulin signaling pathway resistance, due in part to chronic activation of mTOR/S6K1 signaling that results in negative feedback of the insulin signaling pathway and a loss of Akt phosphorylation and insulin content. We evaluated the effects of a new generation insulin sensitizer, MSDC-0160, on restoring insulin/IGF-1 sensitivity and insulin content in human ß-cells. This novel TZD has low affinity for binding and activation of PPARγ and has insulin-sensitizing effects in mouse models of diabetes and ability to lower glucose in Phase 2 clinical trials. MSDC-0160 treatment of human islets increased AMPK activity and reduced mTOR activity. This was associated with the restoration of IGF-1-induced phosphorylation of Akt, GSK-3, and increased protein expression of Pdx1. Furthermore, MSDC-0160 in combination with IGF-1 and 8 mM glucose increased ß-cell specific gene expression of insulin, pdx1, nkx6.1, and nkx2.2, and maintained insulin content without altering glucose-stimulated insulin secretion. Human islets were unable to simultaneously promote DNA synthesis and maintain the ß-cell phenotype. Lithium-induced GSK-3 inhibition that promotes DNA synthesis blocked the ability of MSDC-0160 to maintain the ß-cell phenotype. Conversely, MSDC-0160 prevented an increase in DNA synthesis by blocking ß-catenin nuclear translocation. Due to the counteracting pathways involved in these processes, we employed a sequential ex vivo strategy to first induce human islet DNA synthesis, followed by MSDC-0160 to promote the ß-cell phenotype and insulin content. This new generation PPARγ sparing insulin sensitizer may provide an initial tool for relieving inherent human islet insulin signaling pathway resistance that is necessary to preserve the ß-cell phenotype during ß-cell expansion for the treatment of diabetes.

Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT)--relationship to newly identified mitochondrial pyruvate carrier proteins.

Thiazolidinedione (TZD) insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44) and BRP44 Like (BRP44L), which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT) cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13)C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT) and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.

Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC50 for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data (i) report that clinically used TZDs inhibit the MPC, (ii) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, (iii) indicate that the acute effect of TZDs may be related to insulin sensitization, and (iv) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction.

Insulin resistance and metabolic derangements in obese mice are ameliorated by a novel peroxisome proliferator-activated receptor γ-sparing thiazolidinedione.

Currently approved thiazolidinediones (TZDs) are effective insulin-sensitizing drugs that may have efficacy for treatment of a variety of metabolic and inflammatory diseases, but their use is limited by side effects that are mediated through ectopic activation of the peroxisome proliferator-activated receptor γ (PPARγ). Emerging evidence suggests that the potent anti-diabetic efficacy of TZDs can be separated from the ability to serve as ligands for PPARγ. A novel TZD analog (MSDC-0602) with very low affinity for binding and activation of PPARγ was evaluated for its effects on insulin resistance in obese mice. MSDC-0602 treatment markedly improved several measures of multiorgan insulin sensitivity, adipose tissue inflammation, and hepatic metabolic derangements, including suppressing hepatic lipogenesis and gluconeogenesis. These beneficial effects were mediated at least in part via direct actions on hepatocytes and were preserved in hepatocytes from liver-specific PPARγ(-/-) mice, indicating that PPARγ was not required to suppress these pathways. In conclusion, the beneficial pharmacology exhibited by MSDC-0602 on insulin sensitivity suggests that PPARγ-sparing TZDs are effective for treatment of type 2 diabetes with reduced risk of PPARγ-mediated side effects.

Thiazolidinediones inhibit the progression of established hypertension in the Dahl salt-sensitive rat.

We evaluated the effects of two thiazolidinediones (TZDs), the potent PPARgamma agonist rosiglitazone currently being used to treat diabetes, and a structurally similar experimental compound that is a poor PPARgamma agonist, in a non-diabetic, established hypertension model with continuous measurement of blood pressure by telemetry. Hypertension was induced in male Dahl salt-sensitive rats by a three-week pre-treatment with 4% salt before initiation of treatment. Fasting blood samples were taken for analysis of a biomarker panel to assess metabolic, anti-inflammatory and antioxidant activity of the treatments. Both TZDs significantly reduced both systolic and diastolic blood pressure. When used at the maximally effective doses established for metabolic improvement, both compounds produced equivalent reduction in lipids and elevation of adiponectin, yet the poorer PPARgamma agonist produced significantly greater reductions in blood pressure. Neither compound had a significant effect on circulating glucose or insulin in this animal model. The data demonstrate that these TZDs lower blood pressure significantly in Dahl rats and that this cardiovascular pharmacology is not directly correlated with the metabolic actions or with the magnitude of PPARgamma activation. These data suggest that it may be possible to find insulin-sensitising agents that have beneficial cardiovascular pharmacology with broad applications for disease prevention.

The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria.

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.

Insulin sensitizing pharmacology of thiazolidinediones correlates with mitochondrial gene expression rather than activation of PPAR gamma.

Insulin sensitizing thiazolidinediones (TZDs) are generally considered to work as agonists for the nuclear receptor peroxisome proliferative activated receptor-gamma (PPAR gamma). However, TZDs also have acute, non-genomic metabolic effects and it is unclear which actions are responsible for the beneficial pharmacology of these compounds. We have taken advantage of an analog, based on the metabolism of pioglitazone, which has much reduced ability to activate PPAR gamma. This analog (PNU-91325) was compared to rosiglitazone, the most potent PPAR gamma activator approved for human use, in a variety of studies both in vitro and in vivo. The data demonstrate that PNU-91325 is indeed much less effective than rosiglitazone at activating PPAR gamma both in vitro and in vivo. In contrast, both compounds bound similarly to a mitochondrial binding site and acutely activated PI-3 kinase-directed phosphorylation of AKT, an action that was not affected by elimination of PPAR gamma activation. The two compounds were then compared in vivo in both normal C57 mice and diabetic KKAy mice to determine whether their pharmacology correlated with biomarkers of PPAR gamma activation or with the expression of other gene transcripts. As expected from previous studies, both compounds improved insulin sensitivity in the diabetic mice, and this occurred in spite of the fact that there was little increase in expression of the classic PPAR gamma target biomarker adipocyte binding protein-2 (aP2) with PNU-91325 under these conditions. An examination of transcriptional profiling of key target tissues from mice treated for one week with both compounds demonstrated that the relative pharmacology of the two thiazolidinediones correlated best with an increased expression of an array of mitochondrial proteins and with expression of PPAR gamma coactivator 1-alpha (PGC1 alpha), the master regulator of mitochondrial biogenesis. Thus, important pharmacology of the insulin sensitizing TZDs may involve acute actions, perhaps on the mitochondria, that are independent of direct activation of the nuclear receptor PPAR gamma. These findings suggest a potential alternative route to the discovery of novel insulin sensitizing drugs.

The anatomy and isometry of the medial patellofemoral ligament: implications for reconstruction.

BACKGROUND: Several techniques have been described for reconstruction of the medial patellofemoral ligament. However, the isometry of the medial patellofemoral ligament has not been studied. PURPOSE: To define the anatomy and isometry of the medial patellofemoral ligament. STUDY TYPE: Cadaveric study. METHODS: The authors dissected the medial patellofemoral ligament in 11 cadaveric knees and recorded its anatomic relationships. They evaluated the isometry of the medial patellofemoral ligament by obtaining measurements between various anatomic pairings at certain fixed angles of knee flexion. RESULTS: During knee flexion from 0 degrees to 90 degrees, the portion of the medial patellofemoral ligament from the inferior patellar attachment to the superior femoral attachment was nearly isometric, demonstrating an average change in length of only 1.1 mm. Statistical analysis showed the superior femoral attachment to be most significant in determining isometric behavior. CONCLUSIONS: The most isometric portion of the medial patellofemoral ligament is the inferior portion of its patellar attachment extending to the superior portion of its femoral attachment, with the superior femoral attachment having the most notable influences on isometry. CLINICAL RELEVANCE: This study provides a detailed description of the anatomy of the medial patellofemoral ligament, as well as an evaluation of the isometric behavior of the ligament. Application of these data during reconstruction of the medial patellofemoral ligament may help to optimize patellofemoral stability.

Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe.

Thiazolidinediones address underlying causes of type 2 diabetes, although their mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor-gamma), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for [3H]pioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH2-terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional beta-oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.

Dopamine receptor agonists differ in their actions on cardiac ion channels.

Four dopamine receptor agonists used for the treatment of Parkinson's disease (apomorphine, pergolide, ropinirole and sumanirole) were evaluated for the ability to block human ether-a-go-go related gene (hERG) K(+) channels and to modify the duration of canine Purkinje fiber action potentials. Apomorphine, pergolide and ropinirole blocked the hERG-mediated currents with IC(50) values of 2.4, 0.12 and 1.2 microM, respectively. When evaluated in an action potential duration assay, pergolide significantly shortened action potential duration at 90% repolarization (APD(90)) whereas apomorphine and ropinirole significantly prolonged repolarization. Sumanirole only partially blocked hERG K(+) channels at the highest tested concentration (10 microM) and did not modify action potential duration over the tested concentration range (0.65-65 microM). Taken together, these data provide evidence that dopamine receptor agonists developed for the treatment of Parkinson's disease differentially influence hERG K(+) channel function and cardiac action potential duration.

Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics.

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.