The Fmoc/t-Bu solid-phase synthesis of three difficult peptide sequences (a 9-mer, 15-mer, and 24-mer) was performed using N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on polystyrene, Tentagel, and ChemMatrix resins. In order to obtain an insight into the specific role of the elevated temperature and/or the electromagnetic field for peptide syntheses carried out using microwave irradiation, peptide couplings and Fmoc-deprotection steps were studied under microwave and conventionally heated conditions at the same temperature. While room temperature couplings/deprotections generally produced the difficult peptides in rather poor quality, excellent peptide purities were obtained using microwave heating at a temperature of 86 degrees C for both the coupling and deprotection steps in only 10 and 2.5 min reaction time, respectively. While for most amino acids no significant racemization was observed, the high coupling temperatures led to considerable levels of racemization for the sensitive amino acids His and Cys. It was demonstrated for all three peptide sequences that when performing the coupling/deprotection steps at the same reaction temperature using conventional heating, nearly identical results in terms of both peptide purity and racemization levels were obtained. It therefore appears that the main effect of microwave irradiation applied to solid-phase peptide synthesis is a purely thermal effect not related to the electromagnetic field.

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Mycobacterium tuberculosis is a successful pathogen, and it can survive in infected macrophages in dormant phase for years and decades. The therapy of tuberculosis takes at least six months, and the slow-growing bacterium is resistant to many antibiotics. The development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies is urgently needed. In silico docking methods and structure-based drug design are useful bioinformatics tools for identifying new agents. A docking experiment to M. tuberculosis dUTPase enzyme, which plays a key role in the bacterial metabolism, has resulted in 10 new antitubercular drug candidates. The uptake of antituberculars by infected macrophages is limited by extracellular diffusion. The optimization of the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5 coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H37Rv culture in vitro; the chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5-OT20 tuftsin conjugate exhibited significant antimycobacterial activity on M. tuberculosis H37Rv infected MonoMac6 cells and inhibited intracellular bacteria.

Drug binding induces the disorder-to-order conformational transition of the natively unfolded antimicrobial peptide CM15.

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Tuberculosis (TB) is a bacterial infectious disease caused by Mycobacterium tuberculosis, a slow-growing, powerful human pathogen which can survive in the host macrophages. In the chemotherapy of such intracellular pathogens it is necessary to achieve relatively high level of the drug in blood to attain therapeutically effective concentration in infected cells, which presumably has several serious side effects on healthy tissues. The elimination of M. tuberculosis from infected phagocytes could be more efficient with target cell-directed delivery of antituberculars. A particularly promising approach is to conjugate a drug moiety to a peptide based carrier. The conjugates are chemically constructed to target release by hydrolysis (enzymatic and/or chemical) to liberate the active compound. Here we report the synthesis, characterisation and antimycobacterial evaluation of isoniazid (INH) peptide conjugates. As carrier moiety T-cell epitope of immundominant 16-kDa protein of M. tuberculosis and tuftsin-derived peptides were used. To conjugate INH two synthetic methods were developed, where INH was coupled directly to the peptides or through a heterobifunctional reagent. We found that all of the INH conjugates were effective against M. tuberculosis and the minimal inhibitory concentration (MIC) values were comparable to the free INH moiety.

Considering that Mycobacterium tuberculosis (Mtb) can survive in host phagocytes for decades and currently applied drugs are largely ineffective in killing intracellular Mtb, novel targeted delivery approaches to improve tuberculosis chemotherapy are urgently needed. In order to enhance the efficacy of a clinically used antitubercular agent (isoniazid, INH) a novel lipopeptide carrier was designed based on the sequence of tuftsin, which has been reported as a macrophage-targeting molecule. The conjugate showed relevant in vitro activity on Mtb H37Rv culture with low cytotoxicity and hemolytic activity on human cells. The conjugate directly killed intracellular Mtb and shows much greater efficacy than free INH. To improve bioavailability, the conjugate was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles and tested in vivo in a guinea pig infection model. External clinical signs, detectable mycobacterial colonies in the organs, and the histopathological findings substantiate the potent chemotherapeutic effect of orally administered conjugate-loaded nanoparticles.