Browsing by Author "Ngqinayo, Ntombizanele"

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    The design and synthesis of antituberculosis peptidomimetics focusing on lassomycin derivatives
    (2019) Ngqinayo, Ntombizanele
    Tuberculosis (Tb) is a disease ranked among the top ten causes of death worldwide and is responsible for infecting around 10 million people each year. Tb is caused by the Mycobacterium Tuberculosis (M. tb) bacterial pathogen. The mycobacterium has become resistant towards currently approved drugs which mostly target the cell wall and this has led to the development of the multidrug resistant (MDR) and extremely drug resistant (XDR) M. tb strains. The resistant strains are difficult to treat and require longer treatment duration with the use of combinatory drugs that result in a number of serious side effects. These limitations have led to the search for novel anti-Tb agents and the discovery of lassomycin, an antimicrobial peptide (AMP) that utilizes a different mode of action. The peptide targets the caseinolytic protease of M. tb which is essential for cell survival and causes uncontrolled protein unfolding which results in cell death. Lassomycin is a 16amino acid long basic peptide isolated from a soil bacteria, Lentzea kentuckyensis sp. that been found to be highly selective and potent towards M. tb without affecting mammalian cells.2 The objectives of this project are (i) to modify lassomycin into drug-like derivatives by incorporating N-methylated amino acids to make the peptide more stable against enzymatic degradation; (ii) to shorten the synthetic route by replacing the lactam bridge with a disulfide bridge; (iii) to replace the arginine amino acids in the peptide sequence (difficult to couple) with lysine amino acids to investigate the role of arginine in the binding of the peptide to the acidic region of the caseinolytic enzyme; and (iv) to make the peptide more cationic to improve selectivity for the negatively charged bacterial membrane by adding lysine residues. Peptides were synthesized via the fmoc solid phase peptide synthesis strategy; purified using a semi-preparative High Performance Liquid Chromatogram (prep-HPLC) and analyzed using High Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The bactericidal activity of selected lassomycin derivatives against M. tb was determined using the Alarmar blue assay and one of the derivatives showed a bactericidal effect at a concentration of 9.87 µg/ml which is comparable to that of ethambutol. The derivatives were also found to be selective for pathogens that share a similar protease to that of the M. tb such as Bacillus Subtilis (B. subtilis) and inactive against other pathogens that do not contain the protease. The 3-dimensional structure of the active derivatives will be determined in the future using NMR spectroscopy.
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    The Design, Synthesis and Structure-Activity Relationship of Antitubercular Lassomycin Derivatives
    (University of the Witwatersrand, Johannesburg, 2023) Ngqinayo, Ntombizanele; Makatini, Maya
    Tuberculosis (TB) is a potentially fatal infectious disease caused by Mycobacterium tuberculosis (Mtb) and is a global health risk responsible for over 1.5 million deaths worldwide annually. Tuberculosis is treated with a combinatory regimen of approved first- line drugs such as rifampicin and isoniazid as well as second-line anti-TB drugs such as fluoroquinolones, most of which use similar mechanisms to cause cell death. The formation of multidrug resistance (MDR) TB strains, biofilms, and dormant persister cells (non- replicating cells) are some factors that prolong TB treatment and hence the need for developing novel antitubercular agents with a different mode of action. Furthermore, the emergence of multidrug resistance TB poses a challenge in controlling and eradicating tuberculosis. Lassomycin is a novel antimicrobial peptide (AMP) that has garnered much interest across various research groups due to its ability to effectively target and kill Mtb, including MDR strains and latent TB, with a potency that is similar to that of rifampicin. Lassomycin is highly basic and targets the highly acidic N-terminal domain (NTD) of the caseinolytic enzyme that forms part of the caseinolytic protease crucial for Mtb cell survival. Lassomycin has an unusual mode of action that causes Mtb cell death by disrupting the highly controlled and tightly regulated proteolysis by inhibiting proteolytic activities as well as increasing unfoldase activities. Thus, lassomycin shows great potential as a candidate for drug development. This study aimed to design lassomycin derivatives with improved stability and potency; and synthesize them using shorter and cost-effective synthetic routes. Peptide modifications includes (i) replacing the macrolactam ring in the peptide sequence with a disulfide bridge via a simpler ring-formation method resulting in an enlarged cyclic ring; (ii) replacing ‘difficult’ arginine residues with less basic lysine residues; (iii) forming cationic derivatives by increasing the number of basic lysine residues to enhance selectivity for the bacterial membrane; (iv) conjugating peptide derivatives to lipophilic molecules including palmitic acid and 1-adamantane carboxylic acid to improve bacterial cell penetration and binding; (v) conjugating the peptides to silver nanoparticles for improved drug delivery and antimicrobial effect; (vi) incorporating N-methylated residues to improve peptide stability; (vii) making non-polar peptide derivatives by replacing all basic amino acids with alanine to investigate the importance of the basic residues and study structure activity relationship (SAR) (viii) synthesizing linear derivatives in order to investigate the effect of the ring and (ix) shortening the peptide sequences to include only the cyclic ring or the tail sequence portions in order to shorten the synthetic route. Peptides were synthesized via the Fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis strategy (SPPS) and purified using a semi-preparative High-Performance Liquid Chromatogram (prep-HPLC). They were then analysed using High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS), Circular Dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Silver nanoparticles and the peptide conjugates were characterized using ultraviolet-visible (UV-Vis) spectrophotometry and transmission electron microscopy (TEM) imaging. Two-dimensional (2D) Nuclear magnetic resonance (NMR) spectroscopy, including [1H, 1H] COSY, [1H, 1H] TOCSY, [1H, 13C] HSQC, [1H, 1H] HMBC and [1H, 1H] ROESY were used to determine the structural conformation of Pep-2- NN, a lassomycin derivative that has activity against tuberculosis. Furthermore, the secondary structure of selected derivatives was examined using circular dichroism (CD) spectroscopy. Computational studies were utilized to determine the structure of the active lassomycin derivatives, Pep-2-NN and Pep-2-NNA. All the peptide derivatives were successfully synthesized, including non-polar, short-chained, and those conjugated to silver nanoparticles and lipophilic molecules. The disulfide bridge was successfully added to replace the lactam bridge of the parent lassomycin peptide by oxidising sidechain thiol groups of two cysteine residues inserted at appropriate positions in the sequences. All the peptides were purified to varying degrees of success, and their behaviour was analysed to investigate structure-activity relationships. The silver nanoparticles were successfully synthesized in-house and conjugated to Pep-2-NN. Transmission Electron Microscopy (TEM) imaging revealed that the silver nanoparticles have a spherical morphology at sizes that ranged between 7 nm and 9 nm whilst peptide conjugated nanoparticles were between 9 – 12 nm. Caseinolytic protease (ClpP1P2 or ClpP) assay studies revealed that the peptides display inhibiting and activating properties when screened against the protease, including lassomycin derivatives with shortened chains such as Ring-2-NNA-Ada, Ring-2-NN, and Tail-2-NN. The secondary structure of selected lassomycin derivatives was studied using circular dichroism (CD), revealing that the structures are comprised of anti-parallel beta- (β) sheets at slightly higher proportions followed by alpha- (α) helix and, to some extent, β-turn motif. Computational studies were conducted on selected derivatives to predict their secondary structure and revealed that the peptides form stable α-helical conformations. NMR revealed that Pep-2-NN formed a ‘knotted’ structure, where the tail sequence was threaded inside the cyclic ring with a curved loop, and certain residues in the ring acted as ‘steric plugs’ to prevent unthreading. In conclusion, the insertion of the disulfide bridge remains an effective alternative to the lactam bond found originally on lassomycin and can result in the formation of biologically active derivatives with the desired stable ‘lasso’ conformation.