New pemetrexed‐peptide conjugates: synthesis,characterization and in vitro cytostatic effect on non‐small cell lung carcinoma (NCI‐H358) and human leukemia (HL‐60) cells

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non‐small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem‐peptide conjugates in which cell‐penetrating octaarginine or/and lung‐targeting H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ peptide is attached to the drug by thioether bond. The conjugates characterized by RP‐HPLC and MS exhibited cytostatic effect in vitro on non‐small cell lung carcinoma as well as on human leukemia cell lines. The IC₅₀ values of the conjugates were similar, but the conjugates with H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell‐penetrating or cell‐targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptide conjugates with vitamins for induction of antigen‐specific immunotolerance

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all‐trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen‐specific immunotolerance. We established a synthetic scheme for the preparation of the peptide‐vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen‐specific immunotolerance.     

Isoniazid‐resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase,peroxidase, and INH‐NADH adduct formation activities

Mycobacterium tuberculosis catalase‐peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro‐drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD⁺/NADH forming an isoniazid‐NADH adduct that ultimately confers anti‐tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG‐derived INH‐resistance, we have compared the catalytic properties (including the ability to form the INH‐NADH adduct) of the wild‐type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met‐Tyr‐Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance‐conferring mutants were then assayed for their ability to generate the INH‐NADH adduct in the presence of peroxide (t‐BuOOH and H₂O₂), superoxide, and no exogenous oxidant (air‐only background control). The results demonstrate that residue location plays a critical role in determining INH‐resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant‐specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH‐resistance that is not correlated with the formation of the INH‐NADH adduct.     

Synthesis and antimycobacterial activity of 4-[5-(substituted phenyl)-4, 5-dihydro-3-isoxazolyl]-2-methylphenols

Compounds based on the isoxazoline moiety were screened for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37R (MTB), and INH (isoniazid) resistant Mycobacterium tuberculosis (INHR-MTB) using the agar dilution method and bactec 460. Among the synthesized compounds, 4-[5-(4-bromophenyl)-4,5-dihydro-3-isoxazolyl]-2-methylphenol (4l) was found to be the most active agent against MTB and INHR-MTB with minimum inhibitory concentration of 0.62 μM. When compared to INH, compound (4l) was 1.12 fold and 3.0 fold more active against MTB and INHR-MTB, respectively.     

Iron enhances the susceptibility of pathogenic mycobacteria to isoniazid, an antitubercular drug

The catalase-peroxidase KatG of Mycobacterium tuberculosis plays a central role in the mechanism of action of the anti-tubercular drug isoniazid (INH). Like other bacterial catalases, mycobacterial catalase-peroxidases are dual active enzymes with both catalase and peroxidase activities in the same protein molecule. In our previous study, we showed that iron deprivation resulted in the loss of peroxidase activity in several non-pathogenic mycobacterial species. Here we extended the study to pathogenic mycobacteria and showed that the peroxidase activity, associated with iron-sufficient (4 μg Fe/ml) conditions of growth was responsible for INH activation. Upon iron deprivation (0.02 μg Fe/ml), peroxidase activity was abolished and there was no activation of INH, as demonstrated both by INH-mediated NBT reduction (spectrophotometrically and activity staining in gels) and by viability studies as assayed by the microplate Alamar Blue assay (MABA). In the viability assay, iron-sufficient M. tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG were susceptible to INH and iron-deficient organisms expressing negligible peroxidase survived high concentrations of the drug. It␣is well known that M. tuberculosis is sensitive to low concentrations of INH while the minimum inhibitory concentration of the drug is quite high for other mycobacteria, especially the non-pathogenic species. We showed this difference to be due to the specificity of the peroxidase for the drug. As withholding of iron is one of the host’s mechanisms of controlling an invading pathogen, the implications of these observations on the efficacy of the anti-tubercular drug INH are discussed with reference to the iron status within the human host.     

The katE gene, which encodes the catalase HPII of Mycobacterium avium

Disseminated Mycobacterium avium-Mycobacterium intracellular disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). These pathogens are generally resistant to isoniazid (INH), a powerful antituberculosis drug. It is now generally accepted that the INH susceptibility of Mycobacterium tuberculosis results from the transformation of the drug into a toxic derivative, as a result of the action of the enzyme catalase-peroxidase (HPI), encoded by the katG gene. It has been speculated that the presence of a second catalase (HPII) in some mycobacterial species, but lacking in M. tuberculosis, may impair the action of INH. In this report, the nucleotide sequence of the M. avium katE gene, encoding catalase HPII, is described. This enzyme shows strong similarity to Escherichia coli catalase HPII and eukaryotic catalases. All amino acids previously postulated as participating directly in catalysis by liver catalase and most of the amino acids binding the prosthetic group are conserved in M. avium catalase HPII. The enzyme is expressed in E. coli and is inhibited by 3-amino -l,2,4 triazole (AT). Furthermore, Southern blot hybridizations and polymerase chain reaction experiments demonstrate the distribution of katE gene in several mycobacterial species. To evaluate the potentially antagonistic effect of HPII catalase on INH susceptibility, the katE gene was transformed into M. tuberculosis H37Rv and the minimum inhibitory concentration (MIC) for INH was determined. Despite strong expression of the katEgene, no change in MIC was observed, thus ruling out a possible contribution of this enzyme to the natural resistance of M. avium to the drug. The availability of the gene probe, encoding the second mycobacterial catalase HPII, should open the way for the development of new drugs and diagnostic tests to combat drug-resistant pathogen strains.     

Comparing isogenic strains of Beijing genotype Mycobacterium tuberculosis after acquisition of Isoniazid resistance: A proteomics approach

We determined differences in the protein abundance among two isogenic strains of Mycobacterium tuberculosis (Mtb) with different Isoniazid (INH) susceptibility profiles. The strains were isolated from a pulmonary tuberculosis patient before and after drug treatment. LC‐MS/MS analysis identified 46 Mtb proteins with altered abundance after INH resistance acquisition. Protein abundance comparisons were done evaluating the different bacterial cellular fractions (membrane, cytosol, cell wall and secreted proteins). MS data have been deposited to the ProteomeXchange with identifier PXD002986.     

Isolation of oxidative stress response mutants in Escherichia coli for their use as a genetic screen to identify new anti-mycobacterials as functional analogues of isoniazid

Summary Tuberculosis is a leading killer disease of the world with increasing mortality due to HIV-infected individuals becoming highly prone to this infection. An attempt has been made in the present work to identify novel plant-derived compounds active against Mycobacterium tuberculosis (MTB) through construction of a target based bio-screen to facilitate rapid screening of anti-TB plant compounds. To achieve this, construction of a genetically modified model system was attempted in fast growing, non-pathogenic, Escherichia coli in which experimental testing is relatively easier and rapid as compared to M. tuberculosis, which is pathogenic and slow growing in nature. The exquisitely high sensitivity of M. tuberculosis to isoniazid (INH) has been attributed to lesions in oxyR, a gene that positively regulates the expression of a set of hydrogen peroxide-inducible genes in E. coli and S. typhimurium. Moreover in the mechanism of emergence of INH resistance in M. tuberculosis, oxidative stress response has been implicated. In this study, mutants of E. coli defective in oxidative stress response function were derived and used to screen plant compounds, which might interfere with the oxidative stress response in MTB. Since MTB is inherently known to be oxyR defective and thus being highly sensitive to INH, mutants defective in oxidative stress response were isolated to construct a model system in E. coli, which is otherwise INH resistant, having functional oxyR. These mutants showed simultaneous sensitivity to oxidative stress-causing agents like hydrogen peroxide and cumene hydroperoxide. To further define the mutational lesions, complementation studies were carried out through mobilization of cloned wild type genes involved in the oxidative stress response and in this way a biological screen was constructed to identify plant compounds/essential oils/extracts/oil components which induce oxidative stress. The positives were finally tested for activity against M. tuberculosis strain H37Rv using the radiometric BACTEC 460 TB system. Interestingly, the bioactives were found to be active against the pathogen with marked potency, as the reduction in δGI values for the identified bioactives against M. tuberculosis were significant. The study demonstrates application of a specific target-based genetic model system in E. coli as a rapid high throughput screen in identifying anti-mycobacterials from plants.     

Infection of J774A.1 with different Mycobacterium species induces differential immune and miRNA‐related responses

Macrophages act as a reservoir for Mycobacterium tuberculosis, producing latent infection in approximately 90% of infected people. In this study, J774A.1 mouse macrophage cell line response and microRNA (miRNA) expression during infection with the most relevant mycobacterial strains for humans (M. tuberculosis, M. bovis and M. bovis BCG) was explored. No significant differences in bacillary loads were observed between activate and naive macrophages infected with M. tuberculosis and M. bovis. Nitrite production inhibition and infection control were in accordance with the virulence of the strain. Expression of let‐7e, miR‐21, miR‐155, miR‐210 and miR‐223 was opposite in the two species and miR‐146b* and miR‐1224 expression seemed to be part of the general response to infection.     

Lipase‐catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug‐resistant Mycobacterium tuberculosis strains (M. tuberculosis H₃₇Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435‐catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert‐butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert‐butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88‐99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)‐4‐(1,3‐benzoxazol‐2‐ylsulfanyl)butan‐2‐ol ((±)‐ 3a ), (±)‐4‐[(5‐bromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3b ), and (±)‐4‐[(5,7‐dibromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3c ), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug‐Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50).     

An immunoinformatics approach to define T cell epitopes from polyketide and non‐ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates

The role of polyketide and non‐ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty‐one proteins from polyketide and non‐ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non‐ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T_1/2 ≥ 100 minutes, and 77% of them were mono‐allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I‐peptide complexes. Pathogen peptides that could mimic as self‐peptides or partially self‐peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Synthesis and optimization of hyaluronic acid–methotrexate conjugates to maximize benefit in the treatment of osteoarthritis

We previously reported that a conjugate of hyaluronic acid (HA) and methotrexate (MTX) could be a prototype for future osteoarthritis drugs having the efficacy of the two clinically validated agents but with a reduced risk of the systemic side effects of MTX by using HA as the drug delivery carrier. To identify a clinical candidate, we attempted optimization of a lead, conjugate 1. Initially, in fragmentation experiments with cathepsins, we optimized the peptide part of HA–MTX conjugates to be simpler and more susceptible to enzymatic cleavage. Then we optimized the peptide, the linker, the molecular weight, and the binding ratio of the MTX of the conjugates to inhibit proliferation of human fibroblast-like synoviocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo. Consequently, we found conjugate 30 (DK226) to be a candidate drug for the treatment of osteoarthritis.     

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug‐resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug‐resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug‐resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non‐TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti‐TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti‐mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.     

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis‐infected and culture filtrate protein‐treated macrophages

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC‐MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis‐infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP‐treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis‐infected cells. The exosomes from CFP‐treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.     

Relationship between mutation of serine residue at 315th position in M. tuberculosis catalase-peroxidase enzyme and Isoniazid susceptibility: An in silico analysis

Remarkable advances have been made in the drug therapy of tuberculosis. However much remains to be learned about the molecular and structural basis of drug resistance in Mycobacterium tuberculosis. It is known that, activation of Isoniazid (INH) is mediated by Mycobacterium tuberculosis catalase-peroxidase (MtBKatG) and mutation at position 315 (serine to threonine) leads to resistance. We have conducted studies on the drug resistance through docking and binding analysis supported by time-scale (∼1000 ps) and unrestrained all-atom molecular dynamics simulations of wild and mutant MtBKatG. The study showed conformational changes of binding residues. Mutant (S315T) showed high docking score and INH binding affinity as compared to wild enzyme. In molecular dynamics simulation, mutant enzyme exhibited less structure fluctuation at INH binding residues and more degree of fluctuation at C-terminal domain compared to wild enzyme. Our computational studies and data endorse that MtBKatG mutation (S315T) decrease the flexibility of binding residues and made them rigid by altering the conformational changes, in turn it hampers the INH activity. We ascertain from this work that, this study on structural mechanism of resistance development in Mycobacterium tuberculosis would lead to new therapeutics based on the result obtained in this study.     

Synthesis and mass spectrometry analysis of quaternary cryptando‐peptidic conjugates

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando‐peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando‐peptidic conjugates at the peptide N‐terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision‐induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α‐carbon in the quantitative LC‐MS analysis was analyzed. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis

Aims

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Methods and Results

The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti‐TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4–5 days, the response differences between the sensors made of drug‐sensitive isolates are distinguishable from the sensors made of drug‐resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti‐TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para‐aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions.

Conclusions

To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode‐based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Significance and Impact of the Study

The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O₂ electrode array microbial sensor reactor to enable a high‐throughput drug resistance analysis.