Bioorganic & Medicinal Chemistry Letters 25 (2015) 5825–5830
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Structural measurements and cell line studies of the copper– PEG–Amikacin complex against Mycobacterium tuberculosis Thomas Manning a,⇑, Hatel Patel a, Greg Wylie b, Dennis Phillips c, Jackie Jarvis d a
Chemistry, Valdosta State University, Valdosta, GA 31698, United States Nuclear Magnetic Resonance Facility, Chemistry, University of Georgia, Athens, GA, United States c PAMS Facility, Chemistry, University of Georgia, Athens, GA, United States d FT-ICR Facility, National High Field Magnet Lab, Tallahassee, FL, United States b
a r t i c l e
i n f o
Article history: Received 15 May 2015 Revised 29 July 2015 Accepted 6 August 2015 Available online 8 August 2015 Keywords: Antibiotic Amikacin Resistance Copper PEG Tuberculosis Mycobacterium tuberculosis
a b s t r a c t The bacterium responsible for causing tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) strains. In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, and LC–MS are used to study different aspects of the Copper–polyethylene glycol (PEG)–Amikacin complex. The Cu(II) cation and the aggregate formed by PEG serve as a carrier for the antibiotic. Several Cu–PEG–Amikacin complex variations were tested against NIH-NIAID cell lines containing both resistant and nonresistant strains of M. tuberculosis. Ó 2015 Elsevier Ltd. All rights reserved.
The infectious disease tuberculosis (Tb), caused by the bacterium Mycobacterium tuberculosis, is a major challenge worldwide. An emerging problem in treating Tb is the development of multidrug-resistant tuberculosis strains (MDR-TB).1 When infected aerosols are inhaled into the lungs of a host, tissue dendritic cells and alveolar macrophages respond to M. tuberculosis. This bacterium replicates and survives inside macrophages.2 A group of antibiotics known as aminoglycosides have been used for the past 50 years to treat Tb.1 Aminoglycosides such as Amikacin and Kanamycin are considered second-line medicines used to treat TB and MDR-TB.3–5 Amikacin (see Fig. 1) was originally developed by acetylating a side chain of kanamycin resistant bacteria could bind an inactivating enzyme.4 Amikacin binds to the 16s rRNA in the 30s small ribosomal subunit, which inhibits protein synthesis.6 Amikacin is becoming more prominent in the treatment of tuberculosis due to the propagation of MDR-TB.7 As resistance to tuberculosis becomes a global challenge, second line antibiotics such as Amikacin will continue to emerge. A study by Kumar et al. examined the proteomic analysis of M. tuberculosis isolates resistant to second line drugs (SLDs), and concluded that specific genes and proteins play a role in contributing resistance to these ⇑ Corresponding author. E-mail address: [email protected] (T. Manning). http://dx.doi.org/10.1016/j.bmcl.2015.08.012 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
SLDs.1,6–8 SLDs are highly recognized as a treatment to MDR-TB, but when taken for an extended period, they can cause significant side effects such as nephrotoxicity and ototoxicity. About 19% of MDR-TB patients suffer from hearing loss when given an SLD. Amikacin associated ototoxicity commonly appears even though audio logical assessments are conducted frequently.9 Ototoxicity is associated with a hearing disturbance or vestibular symptoms. Because hearing loss can be irreversible, routine audiograms should be performed in patients to help prevent ototoxicity.10 Utilizing nanoparticles as antibiotic carriers can potentially decrease the toxicity to the body and minimize adverse effects. In some cases, nanoparticles allow for higher control over drug release and encourage specific binding to certain receptors. Therefore, a carrier can be designed to allow a higher percentage of the administered dose to be successful.11 Ghaffari et al. showed that using solid lipid nanoparticles as antibiotic carriers could reduce the amount of Amikacin used by about 50%.12 For this reason, encasing the antibiotic in a polyethylene glycol (PEG) chain was tested. Adding other groups may also increase the efficacy of the antibiotic. Another study has shown that copper (Cu2+) complexes have anti-tuberculosis activity. Copper is necessary for aerobic respiration and creation of certain metalloenzymes, which makes it desirable to the bacteria.13 Typically Tb patients have lower than normal (approximately 1.5–2 ppm)
5826
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
Figure 1. A two-dimensional schematic of the antibiotic Amikacin.
Figure 2. (Top) Amikacin (C22H43N5O13) has a mass of 585.6 g/mol, visible in the spectra, along with its sodium adduct at +23 m/z. (Bottom) the Cu–AMK complex mass spectrum shows no evidence for the free antibiotic (585 m/z) and the sodium adduct (Na–AMK, 608 m/z), spectral features are reduced.
copper levels. The serum concentrations of Fe and Zn are lower and Cu is higher in TB patients. The serum concentration of Zn increased while the serum copper concentration and the copper/ zinc ratio decreased after Tb-antibiotic therapy,14 which suggest the amine containing drugs TB drugs bind copper and remove it from the body when excreted.
Our group has completed several structural and cell line studies involving Fe and Cu complexes with known natural products. Taxol has a single amine which binds to Cu ions (I or II), as can quinine. Quinine has a lower cytotoxicity than taxol against all types of cancer but, along with other anti-malaria drugs, has been shown to have some impact on the spread of pancreatic cancer.15 The Cu
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
5827
Figure 3. The copper–Amikacin isotopic peaks and their natural abundance are 648.215 m/z (51.67%), 649.2 m/z (13.1%), 650.2 m/z (26.2%), 651.2 m/z (6.6%) and 652.2 m/z (1.5%).
Figure 4. (A, top) the proton NMR for the Cu(II)–AMK complex show broadened spectral features due to interaction with the paramagnetic copper ion (B, bottom) the sharp spectral features measured by the 500 MHz proton NMR for the Amikacin complex are evident.
(II)–quinine and hydroxychloroquine complexes are used as a base line for future anti-cancer complexes. In this complex, hydroxychloroquine is utilized for its ability to impact cell autophagy.16,17 The medicinal potential of taxol suffers because of its low water solubility. In another study, binding taxol to Fe(II) or Fe(III) gives it a positive charge, improving its water solubility. However, the iron significantly inhibited its medicinal activity against the National Cancer Institute’s sixty cell line panel.15 Work in this lab has also studied the impact that the copper(II) ion and PEG aggre-
gate approach have on the first and second line Tb drugs Rifmaycin and Capreomycin.18–21 In each case, lower MIC value results in specific experiments provide some optimism that this cationaggregate approach provides some promise as a drug delivery method for antibiotics in the treatment of Tb. Matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is utilized to establish the existence of the Cu–AMK complex. Figure 2 shows the spectral evidence for Amikacin and the sodium adduct of the antibiotics.
5828
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
Figure 3 illustrates the range of 560–630 m/z indicating the parent ion (585 m/z) is absent because the antibiotic is completely complexed to a cation. Figure 3 provides an expanded mass spectral range from 630 to 670 m/z and illustrates the isotopic features that correspond for the Cu–AMK complex. Figure 4A is a sample of a 500 MHz proton NMR spectrum of the copper– Amikacin complex and clearly shows the spectral broadening when compared to Figure 4B, the uncomplexed antibiotic. Using the Irving–Williams series as the precedent,18,19 Cu(II) has a thermodynamic preference for binding to amines when compared to other cations found in a physiological environment such as Zn(II), Ca(II), Mg(II), and Na(I). In addition, Cu(II) can bind oxygen atoms in carbonyl and alcohol functional groups, strengthening its attraction to the pharmaceutical agent. It is important to note that in evaluating the NMR data is the fact that the Cu(II) ion is paramagnetic. The use of NMR in this study is to evaluate mass spectral data and establish that Cu(II) is binding the Amikacin structure. It is important to establish if there is a single site to which the cation binds (i.e., a single amine), or if it is binding at different locations on the molecular structure over time. If it binds at different locations, it would qualify as a polarity adapting molecule, meaning it can change its dipole moment depending on the location of the Cu ion on the molecular structure.20 The proton NMR spectral features in Figures 4 and 5 illustrates that the Cu (II) species mixed with Amikacin at a 1:1 ratio shifts and broadens protons at most locations on the molecule. The changes in the spectral features indicate that the Cu(II) ions are bound and/or
briefly interact at different locations on the structure, indicating that the complex can have a range of polarities. Combining the NMR and MS data signifies that the copper ion binds to the antibiotic, but the species moves around the molecule resulting in various polarities. Figure 6 provides data from Fourier Transform-Ion Cyclotron Resonance (FT-ICR) with an ESI ionization source. FT-ICR provides high resolution and high mass (m/z) accuracy allowing for the assignment of a specific empirical formula. While the MALDITOF-MS does provide information that relates m/z measurements to molar masses, the values are not as accurate as those obtained with FT-ICR. Table 1 provides cell line data for MIC, IC50, and IC90 values measured against M. tuberculosis. For IC50 and IC90 measurements, Cu– PEG–AMK and Cu–PEG results in a slight improvement or lowering of inhibition values. The Cu ion stabilizes the structure, improves the log P values, minimizes chances for hydrogen bonding to unwanted species (Lipinski rules), and allows it to function as a polarity adaptive molecule. While CuCl2 and PEG have no antibacterial activity against M. tuberculosis at lower concentrations, they do increase the efficacy of Amikacin. The detailed reasons for selecting Cu(II) and PEG have been discussed elsewhere18 but to summarize their choices; (i) Cu(II) has a selectivity for binding amines, adds charge and water solubility to the complex and Cu (II) is an essential nutrient so it may be taken in by M. tuberculosis with the drug attached (ii). PEG is a water soluble polymer that forms an aggregate and traps the Amikacin molecule or Cu–Amikacin
Figure 5. A broadening and shifting of spectral feature in the Cu–AMK complex (top) compared to the sharp spectral features shown in the proton NMR of the pure antibiotic (bottom).
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
5829
Figure 6. FT-ICR spectral data for Amikacin and Cu–Amikacin structure are shown, along with the empirical formula assignments.
Table 1 First round IC50, IC90, and MIC values against M. tuberculosis for different complexes Species
IC50 (lM)
IC90 (lM)
MIC (lM)
Amikacin Cu(II)–AMK Cu(II)–PEG AMK–PEG Cu(II)–PEG–AMK
0.216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Structural measurements and cell line studies of the copper– PEG–Amikacin complex against Mycobacterium tuberculosis Thomas Manning a,⇑, Hatel Patel a, Greg Wylie b, Dennis Phillips c, Jackie Jarvis d a
Chemistry, Valdosta State University, Valdosta, GA 31698, United States Nuclear Magnetic Resonance Facility, Chemistry, University of Georgia, Athens, GA, United States c PAMS Facility, Chemistry, University of Georgia, Athens, GA, United States d FT-ICR Facility, National High Field Magnet Lab, Tallahassee, FL, United States b
a r t i c l e
i n f o
Article history: Received 15 May 2015 Revised 29 July 2015 Accepted 6 August 2015 Available online 8 August 2015 Keywords: Antibiotic Amikacin Resistance Copper PEG Tuberculosis Mycobacterium tuberculosis
a b s t r a c t The bacterium responsible for causing tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) strains. In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, and LC–MS are used to study different aspects of the Copper–polyethylene glycol (PEG)–Amikacin complex. The Cu(II) cation and the aggregate formed by PEG serve as a carrier for the antibiotic. Several Cu–PEG–Amikacin complex variations were tested against NIH-NIAID cell lines containing both resistant and nonresistant strains of M. tuberculosis. Ó 2015 Elsevier Ltd. All rights reserved.
The infectious disease tuberculosis (Tb), caused by the bacterium Mycobacterium tuberculosis, is a major challenge worldwide. An emerging problem in treating Tb is the development of multidrug-resistant tuberculosis strains (MDR-TB).1 When infected aerosols are inhaled into the lungs of a host, tissue dendritic cells and alveolar macrophages respond to M. tuberculosis. This bacterium replicates and survives inside macrophages.2 A group of antibiotics known as aminoglycosides have been used for the past 50 years to treat Tb.1 Aminoglycosides such as Amikacin and Kanamycin are considered second-line medicines used to treat TB and MDR-TB.3–5 Amikacin (see Fig. 1) was originally developed by acetylating a side chain of kanamycin resistant bacteria could bind an inactivating enzyme.4 Amikacin binds to the 16s rRNA in the 30s small ribosomal subunit, which inhibits protein synthesis.6 Amikacin is becoming more prominent in the treatment of tuberculosis due to the propagation of MDR-TB.7 As resistance to tuberculosis becomes a global challenge, second line antibiotics such as Amikacin will continue to emerge. A study by Kumar et al. examined the proteomic analysis of M. tuberculosis isolates resistant to second line drugs (SLDs), and concluded that specific genes and proteins play a role in contributing resistance to these ⇑ Corresponding author. E-mail address: [email protected] (T. Manning). http://dx.doi.org/10.1016/j.bmcl.2015.08.012 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
SLDs.1,6–8 SLDs are highly recognized as a treatment to MDR-TB, but when taken for an extended period, they can cause significant side effects such as nephrotoxicity and ototoxicity. About 19% of MDR-TB patients suffer from hearing loss when given an SLD. Amikacin associated ototoxicity commonly appears even though audio logical assessments are conducted frequently.9 Ototoxicity is associated with a hearing disturbance or vestibular symptoms. Because hearing loss can be irreversible, routine audiograms should be performed in patients to help prevent ototoxicity.10 Utilizing nanoparticles as antibiotic carriers can potentially decrease the toxicity to the body and minimize adverse effects. In some cases, nanoparticles allow for higher control over drug release and encourage specific binding to certain receptors. Therefore, a carrier can be designed to allow a higher percentage of the administered dose to be successful.11 Ghaffari et al. showed that using solid lipid nanoparticles as antibiotic carriers could reduce the amount of Amikacin used by about 50%.12 For this reason, encasing the antibiotic in a polyethylene glycol (PEG) chain was tested. Adding other groups may also increase the efficacy of the antibiotic. Another study has shown that copper (Cu2+) complexes have anti-tuberculosis activity. Copper is necessary for aerobic respiration and creation of certain metalloenzymes, which makes it desirable to the bacteria.13 Typically Tb patients have lower than normal (approximately 1.5–2 ppm)
5826
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
Figure 1. A two-dimensional schematic of the antibiotic Amikacin.
Figure 2. (Top) Amikacin (C22H43N5O13) has a mass of 585.6 g/mol, visible in the spectra, along with its sodium adduct at +23 m/z. (Bottom) the Cu–AMK complex mass spectrum shows no evidence for the free antibiotic (585 m/z) and the sodium adduct (Na–AMK, 608 m/z), spectral features are reduced.
copper levels. The serum concentrations of Fe and Zn are lower and Cu is higher in TB patients. The serum concentration of Zn increased while the serum copper concentration and the copper/ zinc ratio decreased after Tb-antibiotic therapy,14 which suggest the amine containing drugs TB drugs bind copper and remove it from the body when excreted.
Our group has completed several structural and cell line studies involving Fe and Cu complexes with known natural products. Taxol has a single amine which binds to Cu ions (I or II), as can quinine. Quinine has a lower cytotoxicity than taxol against all types of cancer but, along with other anti-malaria drugs, has been shown to have some impact on the spread of pancreatic cancer.15 The Cu
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
5827
Figure 3. The copper–Amikacin isotopic peaks and their natural abundance are 648.215 m/z (51.67%), 649.2 m/z (13.1%), 650.2 m/z (26.2%), 651.2 m/z (6.6%) and 652.2 m/z (1.5%).
Figure 4. (A, top) the proton NMR for the Cu(II)–AMK complex show broadened spectral features due to interaction with the paramagnetic copper ion (B, bottom) the sharp spectral features measured by the 500 MHz proton NMR for the Amikacin complex are evident.
(II)–quinine and hydroxychloroquine complexes are used as a base line for future anti-cancer complexes. In this complex, hydroxychloroquine is utilized for its ability to impact cell autophagy.16,17 The medicinal potential of taxol suffers because of its low water solubility. In another study, binding taxol to Fe(II) or Fe(III) gives it a positive charge, improving its water solubility. However, the iron significantly inhibited its medicinal activity against the National Cancer Institute’s sixty cell line panel.15 Work in this lab has also studied the impact that the copper(II) ion and PEG aggre-
gate approach have on the first and second line Tb drugs Rifmaycin and Capreomycin.18–21 In each case, lower MIC value results in specific experiments provide some optimism that this cationaggregate approach provides some promise as a drug delivery method for antibiotics in the treatment of Tb. Matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is utilized to establish the existence of the Cu–AMK complex. Figure 2 shows the spectral evidence for Amikacin and the sodium adduct of the antibiotics.
5828
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
Figure 3 illustrates the range of 560–630 m/z indicating the parent ion (585 m/z) is absent because the antibiotic is completely complexed to a cation. Figure 3 provides an expanded mass spectral range from 630 to 670 m/z and illustrates the isotopic features that correspond for the Cu–AMK complex. Figure 4A is a sample of a 500 MHz proton NMR spectrum of the copper– Amikacin complex and clearly shows the spectral broadening when compared to Figure 4B, the uncomplexed antibiotic. Using the Irving–Williams series as the precedent,18,19 Cu(II) has a thermodynamic preference for binding to amines when compared to other cations found in a physiological environment such as Zn(II), Ca(II), Mg(II), and Na(I). In addition, Cu(II) can bind oxygen atoms in carbonyl and alcohol functional groups, strengthening its attraction to the pharmaceutical agent. It is important to note that in evaluating the NMR data is the fact that the Cu(II) ion is paramagnetic. The use of NMR in this study is to evaluate mass spectral data and establish that Cu(II) is binding the Amikacin structure. It is important to establish if there is a single site to which the cation binds (i.e., a single amine), or if it is binding at different locations on the molecular structure over time. If it binds at different locations, it would qualify as a polarity adapting molecule, meaning it can change its dipole moment depending on the location of the Cu ion on the molecular structure.20 The proton NMR spectral features in Figures 4 and 5 illustrates that the Cu (II) species mixed with Amikacin at a 1:1 ratio shifts and broadens protons at most locations on the molecule. The changes in the spectral features indicate that the Cu(II) ions are bound and/or
briefly interact at different locations on the structure, indicating that the complex can have a range of polarities. Combining the NMR and MS data signifies that the copper ion binds to the antibiotic, but the species moves around the molecule resulting in various polarities. Figure 6 provides data from Fourier Transform-Ion Cyclotron Resonance (FT-ICR) with an ESI ionization source. FT-ICR provides high resolution and high mass (m/z) accuracy allowing for the assignment of a specific empirical formula. While the MALDITOF-MS does provide information that relates m/z measurements to molar masses, the values are not as accurate as those obtained with FT-ICR. Table 1 provides cell line data for MIC, IC50, and IC90 values measured against M. tuberculosis. For IC50 and IC90 measurements, Cu– PEG–AMK and Cu–PEG results in a slight improvement or lowering of inhibition values. The Cu ion stabilizes the structure, improves the log P values, minimizes chances for hydrogen bonding to unwanted species (Lipinski rules), and allows it to function as a polarity adaptive molecule. While CuCl2 and PEG have no antibacterial activity against M. tuberculosis at lower concentrations, they do increase the efficacy of Amikacin. The detailed reasons for selecting Cu(II) and PEG have been discussed elsewhere18 but to summarize their choices; (i) Cu(II) has a selectivity for binding amines, adds charge and water solubility to the complex and Cu (II) is an essential nutrient so it may be taken in by M. tuberculosis with the drug attached (ii). PEG is a water soluble polymer that forms an aggregate and traps the Amikacin molecule or Cu–Amikacin
Figure 5. A broadening and shifting of spectral feature in the Cu–AMK complex (top) compared to the sharp spectral features shown in the proton NMR of the pure antibiotic (bottom).
T. Manning et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5825–5830
5829
Figure 6. FT-ICR spectral data for Amikacin and Cu–Amikacin structure are shown, along with the empirical formula assignments.
Table 1 First round IC50, IC90, and MIC values against M. tuberculosis for different complexes Species
IC50 (lM)
IC90 (lM)
MIC (lM)
Amikacin Cu(II)–AMK Cu(II)–PEG AMK–PEG Cu(II)–PEG–AMK
0.216
