Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2'-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 µl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dH2O. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.

4-Aryl-1,4-Dihydropyridines as Potential Enoyl-Acyl Carrier Protein Reductase Inhibitors: Antitubercular Activity and Molecular Docking Study.

BACKGROUND: Tuberculosis remains one of the most deadly infectious diseases worldwide due to the emergence of multi-drug resistance (MDR) and extensively drug resistance (XDR) strains of Mycobacterium tuberculosis (MTB). AIMS: Currently, available drugs are getting resistant and toxic. Hence, there is an urgent need for the development of potent molecules to treat tuberculosis. MATERIALS AND METHODS: Herein, the screening of a total of eight symmetrical 1,4-dihydropyridine (1,4- DHP) derivatives (4a-4h) was carried out for whole-cell anti-TB activity against the susceptible H37Rv and MDR strains of MTB. RESULTS AND DISCUSSION: Most of the compounds exhibited moderate to excellent activity against the susceptible H37Rv. Moreover, the most promising compound 4f (against H37Rv) having paratrifluoromethyl phenyl group at 4-position and bis para-methoxy benzyl ester group at 3- and 5- positions of 1,4-dihydropyridine pharmacophore, exhibited no toxicity, but demonstrated weak activity against MTB strains resistant to isoniazid and rifampicin. In light of the inhibitory profile of the title compounds, enoyl-acyl carrier protein reductase (InhA) appeared to be the appropriate molecular target. A docking study of these derivatives against InhA receptor revealed favorable binding interactions. Further, in silico predicted ADME properties of these compounds 4a-4h were found to be in the acceptable ranges, including satisfactory Lipinski's rule of five, thereby indicating their potential as drug-like molecules. CONCLUSION: In particular, the 1,4-DHP derivative 4f can be considered an attractive lead molecule for further exploration and development of more potent anti-TB agents as InhA inhibitors.

Structural modification of a novel inhibitor for mycobacterium enoyl-acyl carrier protein reductase assisted by In silico structure-based drug screening.

Background: Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (mtInhA) is involved in the biosynthesis of mycolic acids, a major component of mycobacterial cell walls, and has been targeted in the development of anti-tuberculosis (TB) drugs. In our previous in silico structure-based drug screening study, we identified KES4, a novel class of mtInhA inhibitor. KES4 is composed of four ring structures (A-D-rings) and molecular dynamic simulation predicted that the D-ring is essential for the interaction with mtInhA. Methods: The structure-activity relationship study of the D-ring was attempted and aided by in silico docking simulations to improve the mtInhA inhibitory activity of KES4. A virtual chemical library of the D-ring-modified KES4 was then constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. The candidate compound showing the highest GOLD score, referred to as KEN1, was synthesized, and its biological properties were compared with those of the lead compound KES4. Results: We achieved the synthesis of KEN1 and evaluated its effects on InhA activity, mycobacterial growth, and cytotoxicity. The antimycobacterial activity of KEN1 was comparable to that of the lead compound (KES4), although it exhibited superior activity in mtInhA inhibition. \. Conclusions: We obtained a KES4 derivative with high mtInhA inhibitory activity by in silico docking simulation with a chemical library consisting of a series of D-ring-modified KES4.

Identification of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase inhibitors: A combined in-silico and in-vitro analysis.

Mycobacterium tuberculosis (Mtb), had developed evolutionary changes in its genome to adapt for survival and thereby generated multi-drug resistant strains. However, novel drug targets that remained unchanged for their biochemical role has impressed the research community to target such proteins. The comprehensive analysis of multiple protein targets has influenced us to make a consensus structural rule exploited by pharmacophore and other allied techniques from a large repository of protein structures. In this pursuit, we made a retrospective analysis of pharmacophores mapped from the tuberculosis structural proteome and identified unique patterns that can be employed for the novel molecules design. The current work on NADH-dependent enoyl-acyl carrier protein reductase (InhA) has yielded top scored pharmacophore models which were searched over SPECS natural product database to prioritize the molecules that can be targeted against Mtb. With efforts on rigorous validation and expertise, we have identified such pharmacophoric patterns from natural compounds that can be used as initial hits. Subsequently, these hits were subjected to in-vitro antitubercular evaluation to ensure the inhibitory activity against the mycobacterium culture growth (MtbH37Rv). Furthermore, docking simulations were carried out to provide an insight on the possible modes of interaction between the experimentally explored compounds and InhA.

Antimicrobial activity of antisense peptide-peptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms.

BACKGROUND: Antisense peptide nucleic acids (PNAs) are synthetic polymers that mimic DNA/RNA and inhibit bacterial gene expression in a sequence-specific manner. METHODS: To assess activity against non-typeable Haemophilus influenzae (NTHi), we designed six PNA-peptides that target acpP, encoding an acyl carrier protein. MICs and minimum biofilm eradication concentrations (MBECs) were determined. Resistant strains were selected by serial passages on media with a sub-MIC concentration of acpP-PNA. RESULTS: The MICs of six acpP-PNA-peptides were 2.9-11 mg/L (0.63-2.5 µmol/L) for 20 clinical isolates, indicating susceptibility of planktonic NTHi. By contrast, MBECs were up to 179 mg/L (40 µmol/L). Compared with one original PNA-peptide (acpP-PNA1-3'N), an optimized PNA-peptide (acpP-PNA14-5'L) differs in PNA sequence and has a 5' membrane-penetrating peptide with a linker between the PNA and peptide. The optimized PNA-peptide had an MBEC ranging from 11 to 23 mg/L (2.5-5 µmol/L), indicating susceptibility. A resistant strain that was selected by the original acpP-PNA1-3'N had an SNP that introduced a stop codon in NTHI0044, which is predicted to encode an ATP-binding protein of a conserved ABC transporter. Deletion of NTHI0044 caused resistance to the original acpP-PNA1-3'N, but showed no effect on susceptibility to the optimized acpP-PNA14-5'L. The WT strain remained susceptible to the optimized PNA-peptide after 30 serial passages on media containing the optimized PNA-peptide. CONCLUSIONS: A PNA-peptide that targets acpP, has a 5' membrane-penetrating peptide and has a linker shows excellent activity against planktonic and biofilm NTHi and is associated with a low risk for induction of resistance.

Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex.

BACKGROUND: Members of the Burkholderia cepacia complex (Bcc) cause considerable morbidity and mortality in patients with chronic granulomatous disease and cystic fibrosis. Many Bcc strains are antibiotic resistant, which requires the exploration of novel antimicrobial approaches, including antisense technologies such as phosphorodiamidate morpholino oligomers (PMOs). METHODS: Peptide-conjugated PMOs (PPMOs) were developed to target acpP, which encodes an acyl carrier protein (AcpP) that is thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models. RESULTS: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (>4 log reduction), whereas a PPMO with a scrambled base sequence (scrambled PPMO) had no effect on growth. Human neutrophils were infected with Burkholderia multivorans and treated with AcpP PPMO. AcpP PPMO augmented killing, compared with neutrophils alone and compared with neutrophils alone plus scrambled PPMO. Mice with chronic granulomatous disease that were infected with B. multivorans were treated with AcpP PPMO, scrambled PPMO, or water at 0, 3, and 6 h after infection. Compared with water-treated control mice, the AcpP PPMO-treated mice showed an approximately 80% reduction in the risk of dying by day 30 of the experiment and relatively little pathology. CONCLUSION: AcpP PPMO is active against Bcc infections in vitro and in vivo.

Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei.

Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.

Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose-response in mice infected with Escherichia coli.

OBJECTIVES: Phosphorodiamidate morpholino oligomers (PMOs) are DNA analogues that inhibit translation by an antisense mechanism. Membrane-penetrating peptides attached to PMOs increase PMO efficacy by enhancing penetration through bacterial membranes. The objectives of these experiments are to demonstrate gene-specific efficacy and establish a dose-response relationship of a peptide-PMO conjugate. METHODS: An 11-base PMO (AcpP) targeted at acpP (an essential gene) of Escherichia coli was synthesized and conjugated with the cell-penetrating peptide RFFRFFRFFRXB (X is 6-aminohexanoic acid and B is beta-alanine). Mice were infected by intraperitoneal (i.p.) injection with K-12 E. coli W3110, and treated i.p. at 15 min and 12 h post-infection with various amounts of AcpP peptide-PMO conjugate, AcpP PMO without attached peptide, scrambled base sequence PMOs or ampicillin. A strain (LT1) of E. coli was constructed by replacing acpP with an allele that has four wobble base substitutions in the region targeted by the PMO. RESULTS: Twelve hours after a single treatment, 30 microg of AcpP peptide-PMO or 3 mg of AcpP PMO reduced bacteraemia by 3 orders of magnitude compared with treatment with water. Neither scrambled base sequence PMO controls nor 30 microg of ampicillin reduced bacteraemia. Two treatments with 30 microg of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 microg at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO > 30 microg showed further reductions in plasma cfu. Survival 48 h after treatment with 2 x 30 microg (3 mg/kg) of AcpP peptide-PMO or 2 x 3 mg (300 mg/kg) of AcpP PMO was 100%, compared with 20% for mice treated with water or scrambled base sequence PMO controls. However, survival was reduced to 75% and 0% for mice treated with 2 x 300 microg and 2 x 1 mg of AcpP peptide-PMO, respectively. A conjugate made from the D-isomeric form of each amino acid was less effective than the L-amino acid equivalent, and required 2 x 300 microg treatments for significant reduction in bacteria and survival. Mice infected with LT1 and treated with AcpP peptide-PMO did not survive and had the same amount of bacteria in the blood as mice treated with water, whereas those treated with 2 x 100 microg of AcpPmut4 peptide-PMO (complementary to the mutated allele) survived, and had a 3 orders of magnitude reduction in bacteria in the blood at 24 h post-infection. CONCLUSIONS: Both AcpP peptide-PMO and AcpP PMO significantly reduced bacteraemia and promoted survival of mice infected with E. coli W3110. The conjugate was about 50-100 times more potent than the PMO without attached peptide. The L-isomeric peptide-PMO was 10 times more potent than the D-isomeric equivalent. The conjugate apparently was toxic at doses > or = 2 x 300 microg/mouse (30 mg/kg). PMOs produced a sequence-specific antibiotic effect and the conjugate had a therapeutic index (toxic dose/effective dose) approximately equal to 10 in a mouse model of infection.

Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis.

OBJECTIVES: Antisense phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA mimics that specifically inhibit gene expression in pure cultures of Escherichia coli. Previously, an 11 base PMO targeted to an essential gene (acpP) for phospholipid biosynthesis was shown to inhibit growth of a pure culture of E. coli AS19, which has an abnormally permeable outer membrane. The objectives of experiments in this report are to show that the AcpP PMO significantly inhibits growth of strain SM105, which has a normal, intact outer membrane, both in pure culture and in infected mice. METHODS: In pure culture, SM105 was grown in rich broth supplemented with 20 muM AcpP PMO, and growth was monitored by optical density and viable cell count. Mice were infected by intraperitoneal injection with a non-lethal inoculum of either E. coli AS19 or SM105. Following infection, mice were treated intraperitoneally with 300 mug of the 11 base antisense PMO targeted to acpP, a scrambled sequence PMO or PBS. RESULTS: Growth of SM105 was slower and viable cells were significantly reduced by up to 61% in pure cultures supplemented with AcpP PMO compared with untreated cultures or cultures supplemented with a scrambled sequence PMO. A single dose of AcpP PMO reduced peritoneal cfu of E. coli AS19 about 39- to 600-fold compared with controls at 2, 7, 13 and 23 h after treatment. The same PMO significantly reduced cfu of E. coli SM105 75% compared with controls at 12 h after treatment. However, there was no difference in cfu at 2, 7 or 24 h. A second dose at 24 h again reduced SM105 cfu about 10-fold by 48 h post-infection. In other experiments with infected mice, multiple doses of AcpP PMO sustained the approximately 10-fold reduction in SM105 cfu at 6, 12 and 24 h post-infection. Compared with equivalent (micromolar) doses of ampicillin, AcpP PMO was significantly more effective at all time points. Specificity of PMO inhibition was shown in other experiments by treating infected mice with a PMO targeted to a non-essential reporter gene for luciferase. A luciferase-specific PMO reduced both the amount and activity of luciferase to the same extent, whereas scrambled PMO had no effect. CONCLUSIONS: An 11 base antisense PMO targeted to acpP significantly inhibited viability of a strain of E. coli with a normal, intact outer membrane both in pure culture and in infected mice. Inhibition by PMOs was sequence-specific.

Acyl carrier protein (ACP) inhibition and other differences between beta-ketoacyl synthase (KAS) I and II.

Escherichia coli beta-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BS(3) [bis(sulphosuccinimidyl) suberate] and surface-enhanced laser desorption/ionization-time-of-flight MS disclosed only monomeric and dimeric forms of KAS II, whereas KAS I also forms higher multimers. The binding affinities for KAS I and KAS II to C(14)-acyl carrier protein (ACP) as well as for C(14)-ACP to KAS I and KAS II were determined. KAS I is sensitive to the ACP released during the transfer reaction, with 50% inhibition at 0.17 microM ACP close to the physiological concentration of ACP (0.13 microM). KAS I and II also differ in carrying out the decarboxylation step of the elongation reaction.