RESUMO
Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.
Assuntos
Sumoilação , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos SCID , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sumoilação/efeitos dos fármacos , Transplante Heterólogo , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.
Assuntos
Fosfatase 3 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 3 de Especificidade Dupla/deficiência , Ativação Plaquetária/fisiologia , Embolia Pulmonar/enzimologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária/efeitos dos fármacos , Embolia Pulmonar/sangue , Trombose/sangue , Trombose/enzimologiaRESUMO
Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.
Assuntos
Acetanilidas/química , Acetanilidas/farmacologia , Fosfatase Alcalina/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Acetanilidas/isolamento & purificação , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Isoformas de Proteínas/química , Sulfonamidas/isolamento & purificaçãoRESUMO
Small molecule modulators of phosphatases can lead to clinically useful drugs and serve as invaluable tools to study functional roles of various phosphatases in vivo. Here, we describe lead discovery strategies for identification of inhibitors and activators of intestinal alkaline phosphatases. To identify isozyme-selective inhibitors and activators of the human and mouse intestinal alkaline phosphatases, ultrahigh throughput chemiluminescent assays, utilizing CDP-Star as a substrate, were developed for murine intestinal alkaline phosphatase (mIAP), human intestinal alkaline phosphatase (hIAP), human placental alkaline phosphatase (PLAP), and human tissue-nonspecific alkaline phosphatase (TNAP) isozymes. Using these 1,536-well assays, concurrent HTS screens of the MLSMR library of 323,000 compounds were conducted for human and mouse IAP isozymes monitoring both inhibition and activation. This parallel screening approach led to identification of a novel inhibitory scaffold selective for murine intestinal alkaline phosphatase. SAR efforts based on parallel testing of analogs against different AP isozymes generated a potent inhibitor of the murine IAP with IC50 of 540 nM, at least 65-fold selectivity against human TNAP, and >185 selectivity against human PLAP.
Assuntos
Ativadores de Enzimas/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Animais , Descoberta de Drogas , Ativação Enzimática , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI/agonistas , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Isoenzimas/antagonistas & inibidores , Camundongos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
The hematopoietic protein tyrosine phosphatase (HePTP) is implicated in the development of blood cancers through its ability to negatively regulate the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Small-molecule modulators of HePTP activity may become valuable in treating hematopoietic malignancies such as T cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). Moreover, such compounds will further elucidate the regulation of MAPKs in hematopoietic cells. Although transient activation of MAPKs is crucial for growth and proliferation, prolonged activation of these important signaling molecules induces differentiation, cell cycle arrest, cell senescence, and apoptosis. Specific HePTP inhibitors may promote the latter and thereby may halt the growth of cancer cells. Here, we report the development of a small molecule that augments ERK1/2 and p38 activation in human T cells, specifically by inhibiting HePTP. Structure-activity relationship analysis, in silico docking studies, and mutagenesis experiments reveal how the inhibitor achieves selectivity for HePTP over related phosphatases by interacting with unique amino acid residues in the periphery of the highly conserved catalytic pocket. Importantly, we utilize this compound to show that pharmacological inhibition of HePTP not only augments but also prolongs activation of ERK1/2 and, especially, p38. Moreover, we present similar effects in leukocytes from mice intraperitoneally injected with the inhibitor at doses as low as 3 mg/kg. Our results warrant future studies with this probe compound that may establish HePTP as a new drug target for acute leukemic conditions.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.
Assuntos
Biomarcadores/metabolismo , Fosfotransferases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sequência de Aminoácidos , Biocatálise , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases/metabolismo , Espectrometria de Massas em TandemRESUMO
PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.
Assuntos
Neoplasias Experimentais/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Técnicas de Silenciamento de Genes , Inativação Gênica , Leucemia Experimental/enzimologia , Leucemia Experimental/genética , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNARESUMO
Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.
RESUMO
Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.
Assuntos
Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Domínio Catalítico/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Biofísica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Piruvato Desidrogenase Quinase de Transferência de Acetil , Transdução de SinaisRESUMO
The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of approximately 21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Trifosfato de Adenosina/metabolismo , Biotinilação/efeitos dos fármacos , Humanos , Cinética , Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Fatores de TempoRESUMO
Bacterial elongation factor Tu (EF-Tu) and EF-Ts are interacting proteins involved in polypeptide chain elongation in protein biosynthesis. A novel scintillation proximity assay for the detection of inhibitors of EF-Tu and EF-Ts, as well as the interaction between them, was developed and used in a high-throughput screen of a chemical library. Several compounds from a variety of chemical series with inhibitory properties were identified, including certain indole dipeptides, benzimidazole amidines, 2-arylbenzimidazoles, N-substituted imidazoles, and N-substituted guanidines. The in vitro activities of these compounds were confirmed in a coupled bacterial transcription-translation assay. Several indole dipeptides were identified as inhibitors of bacterial translation, with compound 2 exhibiting a 50% inhibitory concentration of 14 microM and an MIC for S. aureus ATCC 29213 of 5.6 microg/ml. Structure-activity relationship studies around the dipeptidic indoles generated additional analogs with low micromolar MICs for both gram-negative and gram-positive bacteria. To assess the specificity of antibacterial action, these compounds were evaluated in a metabolic labeling assay with Staphylococcus aureus. Inhibition of translation, as well as limited effects on other macromolecular pathways for some of the analogs studied, indicated a possible contribution from a non-target-based antibacterial mechanism of action.
Assuntos
Antibacterianos/química , Dipeptídeos/química , Dipeptídeos/farmacologia , Indóis/química , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Antibacterianos/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Cocos Gram-Positivos/efeitos dos fármacos , Guanidinas/química , Guanidinas/farmacologia , Imidazóis/química , Imidazóis/farmacologia , Substâncias Macromoleculares/metabolismo , Testes de Sensibilidade Microbiana , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacosRESUMO
Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.