Peptidyl tRNA Hydrolase Is Required for Robust Prolyl-tRNA Turnover in Mycobacterium tuberculosis.

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb's susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.

Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients.

Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2(+) patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression.

Phase I evaluation of seliciclib (R-roscovitine), a novel oral cyclin-dependent kinase inhibitor, in patients with advanced malignancies.

AIM: Phase I study of seliciclib (CYC202, R-roscovitine), an inhibitor of cyclin-dependent kinases 2, 7 and 9, causing cell cycle changes and apoptosis in cancer cells. PATIENTS AND METHODS: This phase I trial aimed at defining the toxicity profile, the maximum tolerated dose (MTD), the recommended phase II dose (RD) and the main pharmacokinetic and pharmacodynamic parameters of oral seliciclib. Three schedules were evaluated: seliciclib given twice daily for 5 consecutive days every 3 weeks (schedule A), for 10 consecutive days followed by 2 weeks off (schedule B) and for 3d every 2 weeks (schedule C). RESULTS: Fifty-six patients received a total of 218 cycles of seliciclib. Dose-Limiting Toxicities (DLT) consisting of nausea, vomiting, asthenia and hypokalaemia occurred at 1600 mg bid for schedule A and in schedule C, DLT of hypokalaemia and asthenia occurred at 1800 mg bid. The evaluation of longer treatment duration in schedule B was discontinued because of unacceptable toxicity at lower doses. Other adverse events included transient serum creatinine increases and liver dysfunctions. Pharmacokinetic data showed that exposure to seliciclib and its carboxylate metabolite increased with increasing dose. Soluble cytokeratin 18 fragments allowed monitoring of seliciclib-induced cell death in the blood of patients treated with seliciclib at doses above 800 mg/d. One partial response in a patient with hepatocellular carcinoma and sustained tumour stabilisations were observed. CONCLUSIONS: The MTD and RD for seliciclib are 1250 mg bid for 5d every 3 weeks and 1600 mg bid for 3d every 2 weeks, respectively.

Targeting the cyclin E-Cdk-2 complex represses lung cancer growth by triggering anaphase catastrophe.

PURPOSE: Cyclin-dependent kinases (Cdk) and their associated cyclins are targets for lung cancer therapy and chemoprevention given their frequent deregulation in lung carcinogenesis. This study uncovered previously unrecognized consequences of targeting the cyclin E-Cdk-2 complex in lung cancer. EXPERIMENTAL DESIGN: Cyclin E, Cdk-1, and Cdk-2 were individually targeted for repression with siRNAs in lung cancer cell lines. Cdk-2 was also pharmacologically inhibited with the reversible kinase inhibitor seliciclib. Potential reversibility of seliciclib effects was assessed in washout experiments. Findings were extended to a large panel of cancer cell lines using a robotic-based platform. Consequences of cyclin E-Cdk-2 inhibition on chromosome stability and on in vivo tumorigenicity were explored as were effects of combining seliciclib with different taxanes in lung cancer cell lines. RESULTS: Targeting the cyclin E-Cdk-2 complex, but not Cdk-1, resulted in marked growth inhibition through the induction of multipolar anaphases triggering apoptosis. Treatment with the Cdk-2 kinase inhibitor seliciclib reduced lung cancer formation in a murine syngeneic lung cancer model and decreased immunohistochemical detection of the proliferation markers Ki-67 and cyclin D1 in lung dysplasia spontaneously arising in a transgenic cyclin E-driven mouse model. Combining seliciclib with a taxane resulted in augmented growth inhibition and apoptosis in lung cancer cells. Pharmacogenomic analysis revealed that lung cancer cell lines with mutant ras were especially sensitive to seliciclib. CONCLUSIONS: Induction of multipolar anaphases leading to anaphase catastrophe is a previously unrecognized mechanism engaged by targeting the cyclin E-Cdk-2 complex. This exerts substantial antineoplastic effects in the lung.

Phase I clinical and pharmacokinetic study of oral sapacitabine in patients with acute leukemia and myelodysplastic syndrome.

PURPOSE: Sapacitabine is an oral deoxycytidine nucleoside analog with a unique mechanism of action that is different from cytarabine. PATIENTS AND METHODS: To define the dose-limiting toxicities (DLT) and maximum-tolerated dose (MTD) of sapacitabine given orally twice daily for 7 days every 3 to 4 weeks, or twice daily for 3 days for 2 weeks (days 1 through 3 and days 8 through 10) every 3 to 4 weeks, in refractory-relapse acute leukemia and myelodysplastic syndrome (MDS). A total of 47 patients were treated in the phase I study that used a classical 3 + 3 design. Sapacitabine was escalated from 75 to 375 mg twice daily for 7 days (n = 35) and from 375 to 475 mg twice daily for 3 days on days 1 through 3 and days 8 through 10. RESULTS: The DLTs with both schedules were gastrointestinal. The MTDs were 375 mg twice daily for 7 days and 425 mg twice daily for 3 days on days 1 through 3 and days 8 through 10. The recommended phase II single-agent dose schedules were 325 mg twice daily for 7 days and 425 mg twice daily for 3 days on days 1 through 3 and days 8 through 10. Responses were observed in 13 patients (28%); four were complete responses, and nine were marrow complete responses. CONCLUSION: Sapacitabine is a new, safely administered, oral deoxycytidine analog that has encouraging activity in leukemia and MDS. Phase II studies are ongoing.

Therapeutic efficacy of seliciclib in combination with ionizing radiation for human nasopharyngeal carcinoma.

PURPOSE: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models. EXPERIMENTAL DESIGN: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated. RESULTS: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G(1) and G(2)-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses. CONCLUSIONS: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G(2)-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.

Synergistic inhibition of ErbB signaling by combined treatment with seliciclib and ErbB-targeting agents.

PURPOSE: The aims of this study were to investigate whether the cyclin-dependent kinase inhibitor seliciclib could synergize with agents that target ErbB receptors and to elucidate the molecular mechanism of the observed synergy. EXPERIMENTAL DESIGN: Synergy between seliciclib and ErbB receptor targeted agents was investigated in various cell lines using the Calcusyn median effect model. The molecular mechanism of the observed synergy was studied in cultured cells, and the combination of seliciclib and the epidermal growth factor receptor (EGFR) inhibitor erlotinib was evaluated in an H358 xenograft model. RESULTS: Seliciclib synergized with the anti-HER2 antibody trastuzumab in a breast cancer cell line, which overexpresses the HER2 receptor, and with the erlotinib analogue AG1478 in non-small cell lung cancer cell lines. In the H358 non-small cell lung cancer cell line, synergy involved decreased signaling from the EGFR, with AG1478 directly inhibiting kinase activity while seliciclib decreased the levels of key components of the receptor signaling pathway, resulting in enhanced loss of phosphorylated extracellular signal-regulated kinase and cyclin D1. The combination of seliciclib and erlotinib was evaluated further in an H358 xenograft and shown to be significantly more active than either agent alone. An enhanced loss of cyclin D1 was also seen in vivo. CONCLUSIONS: This is the first report that investigates combining seliciclib with an EGFR inhibitor. The combination decreased signaling from the EGFR in vitro and in vivo and was effective in cell lines containing either wild-type or mutant EGFR, suggesting that it may expand the range of tumors that respond to erlotinib, and therefore, such combinations are worth exploring in the clinic.

Seliciclib, a cell-cycle modulator that acts through the inhibition of cyclin-dependent kinases.

Seliciclib is an inhibitor of cyclin-dependent kinases 2, 7 and 9. Its primary mechanism of action is the inhibition of transcription, resulting in the selective downregulation of rapidly cycling mRNA transcripts, including Mcl-1 and cyclin D1. It possesses antitumour activity as a single agent and also synergises with a wide range of cytotoxic and targeted drugs. Seliciclib has high oral bioavailability and is in clinical development in a capsule formulation. The clinical dose has been determined in Phase I clinical trials for schedules of 3 - 10 consecutive days per cycle of 2 or 3 weeks duration. Its major clinical toxicities include nausea, vomiting, asthenia, hypokalaemia, elevation of creatinine levels and liver function tests, which are reversible after cessation of dosing. Seliciclib is non-myelosuppressive and does not cause intestinal toxicity. Phase II trials have commenced in non-small cell lung cancer and will be initiated shortly in nasopharyngeal carcinoma.

Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1.

Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from all phases of the cell cycle in tumor cell lines, reduces tumor growth in xenografts in nude mice and is currently in phase II clinical trials. This study investigated the mechanism of cell death in multiple myeloma cells treated with seliciclib. In myeloma cells treated in vitro, seliciclib induced rapid dephosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation at these sites is crucial for RNA polymerase II-dependent transcription. Inhibition of transcription would be predicted to exert its greatest effect on gene products where both mRNA and protein have short half-lives, resulting in rapid decline of the protein levels. One such gene product is the antiapoptotic factor Mcl-1, crucial for the survival of a range of cell types including multiple myeloma. As hypothesized, following the inhibition of RNA polymerase II phosphorylation, seliciclib caused rapid Mcl-1 down-regulation, which preceded the induction of apoptosis. The importance of Mcl-1 was confirmed by short interfering RNA, demonstrating that reducing Mcl-1 levels alone was sufficient to induce apoptosis. These results suggest that seliciclib causes myeloma cell death by disrupting the balance between cell survival and apoptosis through the inhibition of transcription and down-regulation of Mcl-1. This study provides the scientific rationale for the clinical development of seliciclib for the treatment of multiple myeloma.

Seliciclib (CYC202 or R-roscovitine), a small-molecule cyclin-dependent kinase inhibitor, mediates activity via down-regulation of Mcl-1 in multiple myeloma.

Cyclin-dependent kinase (CDK) inhibitors have the potential to induce cell-cycle arrest and apoptosis in cancer cells. Seliciclib (CYC202 or R-roscovitine) is a potent CDK inhibitor currently undergoing phase-2 clinical testing in lung and B-cell malignancies. Here we studied the in vitro cytotoxic activity of seliciclib against multiple myeloma (MM) cells. Our data demonstrate that seliciclib has potent cytotoxicity against MM cells that are both sensitive and resistant to conventional therapy as well as primary MM cells from patients. Cell-cycle and Western blot analysis confirmed apoptosis. Importantly, seliciclib triggered a rapid down-regulation of Mcl-1 transcription and protein expression independent of caspase cleavage. Adherence of MM cells to bone marrow stromal cells (BMSCs) induced increased Mcl-1 expression associated with signal transducer and activator of transcription 3 (STAT3) phosphorylation, which was inhibited in a time- and dose-dependent manner by seliciclib. Furthermore, seliciclib inhibited interleukin 6 (IL-6) transcription and secretion triggered by tumor cell binding to BMSCs. Up-regulation of Mcl-1 expression in cocultures was only partially blocked by neutralizing antibody to IL-6, suggesting alternative mechanisms of Mcl-1 modulation by seliciclib. Finally, combination studies of seliciclib with doxorubicin and bortezomib show in vitro synergism, providing the rationale for testing these drug combinations to improve patient outcome in MM.