Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA^Asp by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.Microcins are small (<10-kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (17). Three microcins, B, C, and J, form a subgroup of posttranslationally modified microcins. Members of this subgroup have highly unusual structures and inhibit cellular enzymes that are validated targets for antibacterial drug development (25). Posttranslationally modified microcins are attractive as drug candidates because of their strong antibacterial action and because virtually limitless numbers of their derivatives can be generated by means of mutation, chemical synthesis, or both. Microcin B (McB), a 43-residue peptide with thiazole and indole rings (13), inhibits DNA gyrase (21). Microcin J, a 21-amino-acid peptide, assumes an unusual threaded lasso structure (2, 23, 27) and inhibits bacterial RNA polymerase (1, 18). The structure of the subject of this study, McC (compound 1) is shown in Fig. ​Fig.1a.1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose α-carboxyl group is covalently linked to adenosine through an N-acyl phosphoramide bond (10, 14). The phosphoramidate of McC is additionally modified by an O-propylamine group (9).Open in a separate windowFIG. 1.Structures and synthesis of McC analogs. (a) Structures of microcin C (compound 1) and its processing product (compound 2). (b) Structures of synthetic McC analogs 7 to 9 and their expected processing products, compounds 4 to 6, which are established inhibitors of AspRS, GluRS, and LeuRS, respectively. (c) Structure of Asp-AMP (compound 3), the natural reaction intermediate of AspRS. Compounds 2 and 4 are nonhydrolyzable analogs of this compound. (d) Synthesis of compounds 7 to 9, which starts from compounds 4 to 6. Hereto the hexapeptide was coupled to the sulfamoyl precursors 4-6 via the coupling agent DIC, followed by removal of the Fmoc protecting group: (i) Fmoc-MRTGNA-OH, HOBt, DIC, DIPEA; (ii) Et₃N/DMF (1:1 [vol/vol]).The passage of McC through the inner layer of the Escherichia coli cell wall is carried out by the YejABEF transporter (19). Once inside the cell, McC is specifically processed by one of the several broad-specificity E. coli cytoplasmic aminopeptidases (12). The product of processing, modified aspartyl-adenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. ​(Fig.1c),1c), the natural reaction intermediate of the tRNA^Asp aminoacylation reaction catalyzed by AspRS. However, because the bond between the α-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNA^Asp aminoacylation, while processed McC has no effect on McC-sensitive cells at concentrations at which intact McC strongly inhibits cell growth. Thus, McC is a Trojan horse inhibitor (22): the peptide part allows McC to enter sensitive cells, where it gets processed, liberating the inhibitory part of the drug.Aminoacyl-tRNA synthetases (aaRSs) carry out the condensation of genetically encoded amino acids with cognate tRNAs. When 1 of the 20 aaRSs present in the cell is inhibited, the corresponding tRNA is not charged. This leads to protein synthesis inhibition and cell growth arrest. In principle, variation of the last amino acid of the McC peptide, the product of the mccA gene, should allow investigators to obtain McC derivatives targeting aaRSs other than AspRS. Unfortunately, the results of systematic structure-activity analyses of the McC peptide revealed that substitutions in the seventh codon of mccA invariably prevented McC production, presumably by interfering with posttranslational modifications of the MccA peptide by the McC maturation enzymes (11). Indeed, in vitro analysis showed that the C-terminal asparagine of MccA is required for the addition of the adenosine moiety by the MccB protein (24).Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of their corresponding aaRSs (5, 20, 26). However, these compounds show low in vivo activities due to limited membrane permeability and the absence of a transporter for these compounds. Here, we show that through chemical attachment of aminoacyl-sulfamoyl adenosines to the first 6 amino acids of the MccA peptide, potent antibacterial agents can be generated. The new compounds share the Trojan horse mechanism of action with McC but target aaRSs specified by the last amino acid of the peptide moiety.     

The Leucine-Responsive Regulatory Protein, Lrp, Modulates Microcin J25 Intrinsic Resistance in Escherichia coli by Regulating Expression of the YojI Microcin Exporter

Many Escherichia coli K-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.     

Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC (2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable amounts. Using this synthesis a number of diverse compounds was synthesized with altered target specificity. Further characteristics of the YejABEF transporters were determined using these compounds.     

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC₅₀ values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).     

Modeling Quasispecies and Drug Resistance in Hepatitis C Patients Treated with a Protease Inhibitor

Telaprevir, a novel hepatitis C virus (HCV) NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients infected with HCV. However, drug-resistant HCV variants were detected in vivo at relatively high frequency a few days after drug administration. Here we use a two-strain mathematical model to explain the rapid emergence of drug resistance in HCV patients treated with telaprevir monotherapy. We examine the effects of backward mutation and liver cell proliferation on the preexistence of the mutant virus and the competition between wild-type and drug-resistant virus during therapy. We also extend the two-strain model to a general model with multiple viral strains. Mutations during therapy only have a minor effect on the dynamics of various viral strains, although they are capable of generating low levels of HCV variants that would otherwise be completely suppressed because of fitness disadvantages. Liver cell proliferation may not affect the pretreatment frequency of mutant variants, but is able to influence the quasispecies dynamics during therapy. It is the relative fitness of each mutant strain compared with wild-type that determines which strain(s) will dominate the virus population. This study provides a theoretical framework for exploring the prevalence of preexisting mutant variants and the evolution of drug resistance during treatment with other HCV protease inhibitors or polymerase inhibitors.     

Bacillus thuringiensis section sign-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects

The crystal proteins, or §-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

The Effect of Sporulation Temperature on the Resistance of Bacillus subtilis to a Chemical Inhibitor

The ability of spores of Bacillus subtilis to germinate at 50° in sublethal concentrations of chlorocresol is related to sporulation temperature as is the resistance of the subsequent outgrowth at 50° to this substance. The degree of germination, age of spores and amount of outgrowth produced are of minor importance in determining resistance of the outgrowth.     

Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer

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Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal,Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis

Hepatitis B and C viruses (HBV and HCV, respectively) are different and distinct viruses, but there are striking similarities in their disease potential. Infection by either virus can cause chronic hepatitis, liver cirrhosis, and ultimately, liver cancer, despite the fact that no pathogenetic mechanisms are known which are shared by the two viruses. Our recent studies have suggested that replication of either of these viruses upregulates a cellular protein called serine protease inhibitor Kazal (SPIK). Furthermore, the data have shown that cells containing HBV and HCV are more resistant to serine protease-dependent apoptotic death. Since our previous studies have shown that SPIK is an inhibitor of serine protease-dependent apoptosis, it is hypothesized that the upregulation of SPIK caused by HBV and HCV replication leads to cell resistance to apoptosis. The evasion of apoptotic death by infected cells results in persistent viral replication and constant liver inflammation, which leads to gradual accumulation of genetic changes and eventual development of cancer. These findings suggest a possibility by which HBV and HCV, two very different viruses, can share a common mechanism in provoking liver disease and cancer.Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are serious worldwide health problems, with more than 500 million people believed to be chronically infected with at least one of these viruses (36). HBV is a DNA virus belonging to the Hepadnaviridae family (21), while HCV is an RNA virus belonging to the Flaviviridae family (7). Despite the fact that they are two very different viruses, they share a common pathology in the ability to cause chronic hepatitis, liver cirrhosis, and ultimately, hepatocellular carcinoma (HCC) (34). It remains unclear why these two viruses, which are fundamentally so different, can both lead to similar disease states and the development of HCC.Numerous studies suggest that in chronic viral hepatitis, the host''s immune system is unable to clear infected cells (34). The persistent viral replication further stimulates liver inflammation, and prolonged inflammation and viral persistence result in a gradual accumulation of genetic changes which can subsequently lead to transformation and development of HCC (3, 13). It is possible that part of this failure of the host to clear infected cells results from an inability to induce apoptosis in these cells. For example, persistent HBV/HCV infection suppresses cytotoxic-T-lymphocyte (CTL)-induced apoptosis (3, 4). Apoptosis, or programmed cell death, plays a critical role in embryonic development, immune system function, and the overall maintenance of tissue homeostasis in multicellular organisms. It is also important in the host''s control of viral infection (4). The execution of the apoptotic program has traditionally been considered the result of the activation of a family of proteases known as caspases. Caspase-dependent cell apoptosis (CDCA) usually initiates by activating caspases 8 and 10 through proteolysis of their proenzymes, which further activates the executioner caspases, such as caspase 3 and caspase 7, resulting in the degradation of chromosomal DNA and cell death (28, 29). Recent evidence, however, has suggested that apoptotic cell death can also be promoted and triggered by serine proteases in a caspase-independent manner (5, 6, 39). Serine protease-dependent cell apoptosis (SPDCA) differs from CDCA in that serine proteases, not caspases, are critical to the apoptotic process (1, 6, 39). Interestingly, certain viral infections have been shown to induce SPDCA (27, 39).Failure of the immune-mediated removal of malignant cells through apoptosis may be due to the upregulation of apoptosis inhibitors in these cells (12, 18). We recently demonstrated that SPDCA can be inhibited by a small, 79-amino-acid protein called serine protease inhibitor Kazal (SPIK) (22). SPIK, which is also known as SPINK1, TATI (tumor-associated trypsin inhibitor), and PSTI (pancreas secretory trypsin inhibitor) (8, 24, 38), was first discovered in the pancreas as an inhibitor of autoactivation of trypsinogen (9). The expression of SPIK in normal tissue is limited or inactivated outside the pancreas, but expression of SPIK is elevated in numerous cancers, such as colorectal tumors, renal cell carcinoma, gastric carcinoma, and intrahepatic cholangiocarcinoma (ICC) (16, 19, 24, 31, 40, 41). It remains unknown, however, what role SPIK may play in cancer formation and development. Additionally, overexpression of SPIK was also found in HBV/HCV-infected human livers (32), and an even higher level of expression of SPIK was found in HBV/HCV-associated HCC tissue (19, 31). This implies that SPIK may be closely associated with hepatitis virus infection and development of HCC.Here we show direct evidence that HBV/HCV replication does in fact upregulate expression of the apoptosis inhibitor SPIK, resulting in resistance to SPDCA, which could ultimately lead to the development of chronic hepatitis and liver cancer.     

Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide

Molecular Biology - The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a...     

Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly¹² to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 amino acid residues (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-G) (4, 12). Four genes (mcjA, mcjB, mcjC, and mcjD) are required for MccJ25 synthesis, export, and immunity (14, 15). The mcjA gene encodes a 58-amino-acid MccJ25 precursor, which is processed by the products of mcjC and mcjB (7). Once synthesized, the mature MccJ25 is excreted to the medium by McjD, an ABC-type transporter (6, 14). The tertiary structure of MccJ25 was elucidated as a lariat peptide (1, 10, 17). It contains an eight-residue ring (G¹ to E⁸) and a tail (Y⁹ to G²¹) whose C-terminal end is sterically trapped within the ring. MccJ25 amino acids F¹⁰ to P¹⁶ form a β-hairpin structure, comprising two β-strands (F¹⁰-V¹¹ and T¹⁵-P¹⁶) and a β-turn (V¹¹ to G¹⁴).MccJ25 is active on gram-negative bacteria related to the producer strain, and among them are several human pathogens (11, 12, 16). It was previously shown that the E. coli RNA polymerase (5, 18) and the bacterial respiratory chain (2, 9) are the targets for MccJ25 action. MccJ25 is active on pathogenic strains of Salmonella spp., Shigella spp. (12), and E. coli, including O157:H7 (11) as well as non-O157 strains (data not shown), that frequently cause outbreaks of food-borne diseases. In addition, Sable et al. (11) showed that MccJ25 was the most active microcin against 12 out of 15 diarrheagenic E. coli strains tested. These authors also demonstrated that MccJ25 inhibits E. coli O157:H7 in biological products such as milk, egg yolk, and meat extract. These findings suggest that MccJ25 could be an efficient complement to nisin for food preservation. However, the potential usefulness of MccJ25 is compromised by the fact that it is highly resistant to digestion by proteolytic enzymes of the stomach (pepsin) and intestinal (trypsin, chymotrypsin, and carboxypeptidases) contents. Thus, the antibiotic would most likely remain active in the intestine, and this could lead to disturbance of the normal microbiota. Therefore, for potential use of MccJ25 as a food additive, it would be desirable to render MccJ25 susceptible to at least one of these proteases. In the present work, we describe a chymotrypsin-susceptible MccJ25 derivative that remains fully active on S. Newport and E. coli O157:H7 in biological products, namely milk and egg yolk. In addition, we demonstrate that the peptide is inactivated by rat intestinal contents.     

Hepatitis C Virus Resistance to Carbohydrate-Binding Agents

Carbohydrate binding agents (CBAs), including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA), Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.     

Compensatory Increase of Transglutaminase 2 Is Responsible for Resistance to mTOR Inhibitor Treatment

The mechanistic target of rapamycin complex 1 (mTORC1) plays a crucial role in controlling cell growth and homeostasis. Deregulation of mTOR signaling is frequently observed in some cancers, making it an attractive drug target for cancer therapy. Although mTORC1 inhibitor rapalog-based therapy has shown positive results in various pre-clinical animal cancer studies, tumors rebound upon treatment discontinuation. Moreover, several recent clinical trials showed that the mTORC1 inhibitors rapamycin and rapalog only reduce the capacity for cell proliferation without promoting cell death, consistent with the concept that rapamycin is cytostatic and reduces disease progression but is not cytotoxic. It is imperative that rapamycin-regulated events and additional targets for more effective drug combinations be identified. Here, we report that rapamycin treatment promotes a compensatory increase in transglutaminase 2 (TGM2) levels in mTORC1-driven tumors. TGM2 inhibition potently sensitizes mTORC1-hyperactive cancer cells to rapamycin treatment, and a rapamycin-induced autophagy blockade inhibits the compensatory TGM2 upregulation. More importantly, tumor regression was observed in MCF-7-xenograft tumor-bearing mice treated with both mTORC1 and TGM2 inhibitors compared with those treated with either a single inhibitor or the vehicle control. These results demonstrate a critical role for the compensatory increase in transglutaminase 2 levels in promoting mTORC1 inhibitor resistance and suggest that rational combination therapy may potentially suppress cancer therapy resistance.