novobiocin and Neoplasms

Hsp90 is one of the most important chaperones involved in regulating the maturation of more than 300 client proteins, many of which are closely associated with refractory diseases, including cancer, neurodegenerative diseases, and viral infections. Clinical Hsp90 inhibitors bind to the ATP pocket in the N-terminal domain of Hsp90 and subsequently suppress the ATPase activity of Hsp90. Recently, with the increased understanding of the discrepancies in the isoforms of Hsp90 and the modes of Hsp90-co-chaperone-client complex interactions, some new strategies for Hsp90 inhibition have emerged. Novel Hsp90 inhibitors that offer selective suppression of Hsp90 isoforms or specific disruption of Hsp90-co-chaperone protein-protein interactions are expected to show with satisfactory efficacy and safety profiles. This review summarizes the recent progress in Hsp90 inhibitors. Additionally, Hsp90 inhibitory strategies are emphasized in this review.

Topics: Animals; Antineoplastic Agents; Autoimmune Diseases; Benzoquinones; Forecasting; HSP90 Heat-Shock Proteins; Humans; Immunosuppressive Agents; Lactams, Macrocyclic; Molecular Chaperones; Neoplasms; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-link

Topics: Adenosine Triphosphate; Animals; Binding Sites; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Neurodegenerative Diseases; Novobiocin; Protein Binding

Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for stability and function of multiple mutated, chimeric, and over-expressed signaling proteins that promote cancer cell growth and/or survival. Hsp90 client proteins include telomerase, mutated p53, Bcr-Abl, Raf-1, Akt, HER2/Neu (ErbB2), mutated B-Raf, mutated EGF receptor, and HIF-1alpha. Hsp90 inhibitors, by interacting specifically with a single molecular target, cause inactivation, destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising anti-tumor activity in various preclinical tumor models. One Hsp90 inhibitor, 17-AAG, is currently in Phase II clinical trial and other inhibitors will shortly be entering the clinic. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple signaling pathways on which cancer cells depend for growth and survival. Identification of benzoquinone ansamycins as the first Hsp90 inhibitors allowed investigators to determine the biologic effects, at first in vitro and then in vivo, of pharmacologic inhibition of Hsp90. These studies rapidly enhanced our understanding of Hsp90 function and led to the identification of radicicol as a structurally distinct Hsp90 inhibitor. Additional target-based screening uncovered novobiocin as a third structurally distinct small molecule with Hsp90 inhibitory properties. Use of novobiocin, in turn, led to identification of a previously uncharacterized C-terminal ATP binding site in the chaperone. Small molecule inhibitors of Hsp90 have been very useful in understanding Hsp90 biology and in validating this protein as a molecular target for anti-cancer drug development.

Topics: Animals; Antibiotics, Antineoplastic; Biological Products; Drug Design; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Macrolides; Molecular Structure; Neoplasms; Novobiocin

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target for cancer therapy. It operates as part of a multichaperone complex and is essential for the conformation, stability, and function of several key oncogenic client proteins such as mutant p53, ERBB2, B-RAF, C-RAF, and CDK4. The HSP90-based chaperone machine is driven by the hydrolysis of ATP and ADP/ATP nucleotide exchange. Many of the inhibitors of HSP90 interrupt the intrinsic ATPase activity, causing degradation of the client proteins via the ubiquitin-proteasome pathway. The first-in-class HSP90 inhibitor in clinical trials is the geldanamycin analog, 17-allylamino, 17-demethoxygeldanamycin (17-AAG). The results that have emerged from these trials have been encouraging, with stable disease observed in two melanoma patients. Pharmacodynamic endpoints, such as induction of HSP70 and downregulation of C-RAF and CDK4 in peripheral blood mononuclear cells and tumor biopsies from treated patients, provided evidence of HSP90 inhibition at well-tolerated doses. The toxicity of 17-AAG has been mild. Several preclinical studies have shown that 17-AAG may enhance the efficacy of a variety of chemotherapeutic agents. Phase II clinical trials in various cancers have been initiated as well as Phase I trials of combined therapy with 17-AAG. However, there are several limitations with 17-AAG such as solubility, stability, and hepatotoxicity. Thus, it is not surprising that new HSP90 agents are under development against this novel target for cancer therapy and several show promise.

Topics: Animals; Antineoplastic Agents; Benzoquinones; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Models, Biological; Models, Chemical; Molecular Chaperones; Neoplasms; Novobiocin; Purines; Pyrazoles

A deregulated activity of PKB/Akt (where PKB stands for protein kinase B) renders tumour cells resistant to a variety of apoptosis-inducing stimuli. Elucidation of the mechanisms responsible for this deregulation is of prime importance for the development of novel anti-cancer drugs. Results of the present study demonstrate that the constitutive activity of PKB/Akt in B16BL6 melanoma cells depends on the integrity of cholesterol-enriched membrane microdomains, since the exposure of cells to cholesterol-depleting agents decreases the phosphorylation of this enzyme, with no change in its total protein level. Inhibitors of Hsp90 (heat-shock protein 90) decreased phosphorylation of PKB/Akt with a similar pattern. Dephosphorylation of the enzyme, as a consequence of raft disintegration, could be precluded by inhibition of serine/threonine (but not tyrosine) phosphatases. Our results imply that destabilization of lipid rafts seemingly affects the association of Hsp90 with the respective serine/threonine phosphatases, thereby increasing the accessibility to PKB/Akt to deactivating phosphatases. We have found recently that reconstituted expression of H-2K class I glycoproteins in class I-deficient B16BL6 cells also decreases the phosphorylation of PKB/Akt. Therefore it is possible that raft-associated regulation of this important enzyme involves both H-2K glycoproteins and Hsp90.

Topics: Animals; Antineoplastic Agents; Cholesterol; Glycoproteins; HSP90 Heat-Shock Proteins; Melanoma, Experimental; Membrane Microdomains; Mice; Neoplasms; Novobiocin; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt

In vertebrates, the glucuronidation of small lipophilic agents is catalyzed by the endoplasmic reticulum UDP-glucuronosyltransferases (UGTs). This metabolic pathway leads to the formation of water-soluble metabolites originating from normal dietary processes, cellular catabolism, or exposure to drugs and xenobiotics. This classic detoxification process, which led to the discovery nearly 50 years ago of the cosubstrate UDP-glucuronic acid (19), is now known to be carried out by 15 human UGTs. Characterization of the individual gene products using cDNA expression experiments has led to the identification of over 350 individual compounds that serve as substrates for this superfamily of proteins. This data, coupled with the introduction of sophisticated RNA detection techniques designed to elucidate patterns of gene expression of the UGT superfamily in human liver and extrahepatic tissues of the gastrointestinal tract, has aided in understanding the contribution of glucuronidation toward epithelial first-pass metabolism. In addition, characterization of the UGT1A locus and genetic studies directed at understanding the role of bilirubin glucuronidation and the biochemical basis of the clinical symptoms found in unconjugated hyperbilirubinemia have uncovered the structural gene polymorphisms associated with Crigler-Najjar's and Gilbert's syndrome. The role of the UGTs in metabolism and different disease states in humans is the topic of this review.

Topics: Autoimmunity; Chromosome Mapping; Glucuronides; Glucuronosyltransferase; Humans; Hyperbilirubinemia; Neoplasms; Steroids; Terminology as Topic

Topics: Chromatin; DNA; DNA Repair; Humans; Karyotyping; Leukemia, Lymphoid; Nalidixic Acid; Neoplasms; Novobiocin; Nucleic Acid Conformation; Plasmids; Recombination, Genetic; Tetradecanoylphorbol Acetate

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors.. Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Topics: DNA; DNA End-Joining Repair; DNA Repair; DNA-Activated Protein Kinase; DNA-Directed DNA Polymerase; Humans; Neoplasms; Novobiocin; Pyridazines; Quinazolines; Synthetic Lethal Mutations; Tumor Suppressor Protein p53

Staphylococcus hominis subsp. novobiosepticus is a new sub-species of S. hominis, thus dividing S. hominis into subsp. hominis and novobiosepticus. This study was designed to identify subsp. novobiosepticus isolates amongst the S. hominis isolated from blood samples of patients with malignancy and septicaemia and to study their resistance profile. The identification was performed by using three simple tests which differentiated between the two sub-species. It was found that 22.8 per cent of S. hominis isolates belonged to subsp. novobiosepticus.

Topics: Acetylglucosamine; Adult; Aged; Drug Resistance, Multiple; Female; Humans; Male; Middle Aged; Neoplasms; Novobiocin; Sepsis; Staphylococcus hominis; Trehalose

Hsp90 is responsible for the conformational maturation of newly synthesized polypeptides (client proteins) and the re-maturation of denatured proteins via the Hsp90 chaperone cycle. Inhibition of the Hsp90 N-terminus has emerged as a clinically relevant strategy for anticancer chemotherapeutics due to the involvement of clients in a variety of oncogenic pathways. Several immunophilins, co-chaperones and partner proteins are also necessary for Hsp90 chaperoning activity. Alternative strategies to inhibit Hsp90 function include disruption of the C-terminal dimerization domain and the Hsp90 heteroprotein complex. C-terminal inhibitors and Hsp90 co-chaperone disruptors prevent cancer cell proliferation similar to N-terminal inhibitors and destabilize client proteins without induction of heat shock proteins. Herein, current Hsp90 inhibitors, the chaperone cycle, and regulation of this cycle will be discussed.

Topics: Animals; Antineoplastic Agents; Catechin; Drug Discovery; HSP90 Heat-Shock Proteins; Humans; Models, Molecular; Neoplasms; Novobiocin; Silybin; Silymarin

The half maximal inhibitory concentration (IC₅₀) has several limitations that make it unsuitable for examining a large number of compounds in cytotoxicity studies, particularly when multiple exposure periods are tested. This article proposes a new approach to measure drug effectiveness, which allows ranking compounds according to their toxic effects on live cells. This effectiveness measure, which combines all exposure times tested, compares the growth rates of a particular cell line in the presence of the compound with its growth rate in the presence of DMSO alone. Our approach allows measuring a wider spectrum of toxicity than the IC₅₀ approach, and allows automatic analyses of a large number of compounds. It can be easily implemented in linear regression software, provides a comparable measure of effectiveness for each investigated compound (both toxic and non-toxic), and allows statistically testing the null hypothesis that a compound is non-toxic versus the alternative that it is toxic. Importantly, our approach allows defining an automated decision rule for deciding whether a compound is significantly toxic. As an illustration, we describe the results of a cellbased study of the cytotoxicity of 24 analogs of novobiocin, a C-terminal inhibitor of heat shock protein 90 (Hsp90); the compounds were ranked in order of cytotoxicity to a panel of 18 cancer cell lines and 1 normal cell line. Our approach may also be a good alternative to computing the half maximal effective concentration (EC₅₀) in studies searching for compounds that promote cell growth.

Topics: Antineoplastic Agents; Cell Line; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; HSP90 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Models, Biological; Models, Statistical; Neoplasms; Novobiocin

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ∼700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure-activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest midnanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.

Topics: Anti-Bacterial Agents; Antineoplastic Agents; Carbohydrates; Cell Line, Tumor; Cell Proliferation; Coumarins; Drug Design; Female; HSP90 Heat-Shock Proteins; Humans; Male; Molecular Structure; Molecular Targeted Therapy; Neoplasms; Novobiocin; Structure-Activity Relationship

Heat shock protein 90 (Hsp90) takes part in the developments of several cancers. Novobiocin, a typically C-terminal inhibitor for Hsp90, will probably used as an important anticancer drug in the future. In this work, we explored the valuable information and designed new novobiocin derivatives based on a three-dimensional quantitative structure-activity relationship (3D QSAR). The comparative molecular field analysis and comparative molecular similarity indices analysis models with high predictive capability were established, and their reliabilities are supported by the statistical parameters. Based on the several important influence factors obtained from these models, six new novobiocin derivatives with higher inhibitory activities were designed and confirmed by the molecular simulation with our models, which provide the potential anticancer drug leads for further research.

Topics: Antineoplastic Agents; Binding Sites; Computer Simulation; Drug Design; HSP90 Heat-Shock Proteins; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Least-Squares Analysis; Models, Molecular; Molecular Structure; Neoplasms; Novobiocin; Protein Binding; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Static Electricity; Thermodynamics

We evaluated whether inhibition of heat shock protein 90 (hsp90) function by novobiocin derivatives could induce the degradation of signal transducers that drive cancer cell growth and thereby promote apoptosis. Removal of the noviose moiety in novobiocin and introduction of a tosyl substituent at C-4 or C-7 coumarin nucleus provided derivatives 4TCNA and 7TCNA which compared favourably with novobiocin in MCF-7 breast cancer cells. Here we extend the antiproliferative and apoptotic properties of these analogues to a panel of cancer cell lines. Destabilization of hsp90 client proteins Raf-1, HER2, and cdk4 suggests inhibition of hsp90 chaperoning function. In HT29 colon and IGROV1 ovarian cancer cells, the growth inhibiting effect of 4TCNA and 7TCNA was consistent with the stimulation of cell death as assessed by the processing and activation of caspase 9, 8, 7 and 3 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP). In Ishikawa endometrial adenocarcinoma cells, 4TCNA also promoted apoptosis and the processing of PARP. These derivatives impacting multiple pathways involved in the neoplastic process may represent promising drugs for cancer therapy.

Topics: Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase 4; Flow Cytometry; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Novobiocin; Poly(ADP-ribose) Polymerases; Tosyl Compounds; Tumor Cells, Cultured

The cochaperone p23 is required for the chaperoning cycle of hsp90 and to enhance the maturation of several client proteins. Tosylcyclonovobiocic acids (4TCNA and 7TCNA) are potent analogs of novobiocin and induce cell cycle arrest, apoptosis and degradation of hsp90 client proteins in a panel of cancer cells. In this study, Western blotting shows that 4TCNA and 7TCNA triggered processing of the hsp90 cochaperone p23 in a dose-dependent manner. Small interfering RNA (siRNA)-mediated reduction of p23 expression in MCF-7 breast cancer cells did not block 4TCNA-induced caspase activation as assessed by the cleavage of PARP. This result indicates that 4TCNA-mediated cell death is a p23-independent process. In HT29 colon cancer cells, 4TCNA and 7TCNA up-regulated GRP78 and GRP94 supporting involvement of ER stress in apoptosis.

Topics: Apoptosis; Cell Line, Tumor; Collagen Type XI; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Estrogen Receptor alpha; Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Intramolecular Oxidoreductases; Membrane Glycoproteins; Molecular Chaperones; Neoplasms; Novobiocin; Prostaglandin-E Synthases; RNA Interference; RNA, Small Interfering; Stress, Physiological; Up-Regulation

The susceptibility of clinical isolates of methicillin-susceptible and -resistant staphylococci from cancer patients with central venous catheter bacteremia to quinupristin/dalfopristin, a semisynthetic streptogramin, was determined in vitro. Susceptibility of these isolates to nine other antistaphylococcal antibiotics was also determined for comparison. A total of 197 staphylococcal strains were tested from 1983 to 1992. Quinupristin/dalfopristin was bactericidal against all isolates, independent of their resistance to methicillin. Its activity was similar to that of vancomycin but superior to that of teicoplanin. Quinupristin/dalfopristin may prove to be an important addition to our armamentarium against catheter-related staphylococcal infections.

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antibiotics, Antitubercular; Bacteremia; Catheterization, Central Venous; Cefamandole; Cephalosporins; Ciprofloxacin; Clindamycin; Daptomycin; Humans; Methicillin; Methicillin Resistance; Microbial Sensitivity Tests; Neoplasms; Novobiocin; Oxacillin; Penicillins; Rifampin; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Teicoplanin; Vancomycin; Virginiamycin

Antineoplastic drug resistance is a major obstacle to improved treatment of most adult cancers in humans. Novobiocin, an antibacterial agent which inhibits the eukaryotic topoisomerase II enzyme, increases the cytotoxicity of several alkylating agents in vitro by the formation of lethal DNA-DNA interstrand cross-links, perhaps by decreasing the repair of drug monoadducts. In murine tumors treated in vivo novobiocin markedly potentiates alkylating agent cytotoxicity without concomitant increases in host toxicity. With this background, a Phase I trial of novobiocin and cyclophosphamide was performed in refractory cancer patients. Novobiocin was given p.o. for 96 h; 750 mg/m2 of i.v. cyclophosphamide was administered at 48 h. Thirty-four patients received 65 courses. The dose-limiting toxicity of novobiocin in this trial was vomiting. The maximum tolerated dose was 6 g/day. Six of 34 patients had Grade III or IV mylosuppression but no dose escalation effect was noted. Three patients developed allergic reactions which resolved completely. No other significant toxicity occurred. While no dose-dependent effect on serum novobiocin levels occurred, 18 of 19 patients treated at greater than or equal to 4 g daily had serum levels greater than or equal to 100 micrograms/ml at steady state, a level which corresponds to levels used in vitro and seen in vivo where the murine novobiocin half-life of 82 min is far less than that seen in humans (6.0 h). Two of 30 evaluable patients had partial responses. Four other patients had stable disease. Four of six had prior disease progression on cyclophosphamide combination therapy. Novobiocin is well tolerated in patients receiving cyclophosphamide and blood levels are in the drug-potentiating range. Phase II trials in cyclophosphamide refractory patients are anticipated.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Drug Evaluation; Female; Humans; Male; Mice; Mice, Inbred C3H; Neoplasms; Novobiocin; Sarcoma, Experimental

Comparisons have been made of the DNA polymerases of normal human lung and cecum, primary carcinomas of human lung, breast, and cecum, and resting and regeneration rat liver. The picture for the normal human tissues is similar to the one for unstimulated rat liver, that for the human carcinomas resembles regenerating rat liver. The human tissues contain two polymerases with sedimentation coefficients of about 3 and 7 S, the enzymes are restricted to the nucleus, and the specific activities of the 7 S polymerase, but not of the 3 S enzyme, are elevated in the cancers. Just as with the regenerating rat liver polymerases, the 3 S activity of a bronchogenic carcinoma is unaffected by cytosine arabinoside 5'-triphosphate and only little reduced by novobiocin, whereas DNA synthesis by the 7 S enzyme is abolished by both compounds. A variety of other inhibitory agents have similar effects on the 7 S polymerases of the human carcinomas and regenerating rat liver.

Topics: Animals; Breast Neoplasms; Cecal Neoplasms; Cecum; Cell Nucleus; Cytarabine; DNA-Directed DNA Polymerase; DNA, Neoplasm; Humans; Isoenzymes; Liver; Liver Regeneration; Lung; Lung Neoplasms; Neoplasms; Novobiocin; Nucleic Acid Synthesis Inhibitors; Rats

Topics: Ampicillin; Anti-Bacterial Agents; Carbenicillin; Chloramphenicol; Colistin; Drug Combinations; Drug Interactions; Drug Synergism; Enterobacteriaceae Infections; Erythromycin; Gentamicins; Glycosides; Humans; Hydrogen-Ion Concentration; Infections; Neoplasms; Novobiocin; Penicillins; Polymyxins; Streptomycin; Sulfonamides; Tetracycline; Trimethoprim