Specific interactions between rifamycin antibiotics and water

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Specific interactions between rifamycin antibiotics and water influencing ability to overcome natural cell barriers and the range of antibacterial potency Krystian Pyta, Anna Janas, Natalia Skrzypczak, Wojciech Schilf, Barbara Wicher, Maria Gdaniec, Franz Bartl, and Piotr Przybylski ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00176 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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ACS Infectious Diseases

Specific interactions between rifamycin antibiotics and water influencing ability to overcome natural cell barriers and the range of antibacterial potency Krystian Pyta,a Anna Janas,a Natalia Skrzypczak,a Wojciech Schilf,b Barbara Wicher,c Maria Gdaniec,a Franz Bartl,d Piotr Przybylski*a aFaculty

of Chemistry, Adam Mickiewicz University, Uniwersytetu Poznanskiego 8, 61-614 Poznan, Poland

bInstitute

of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland

cDepartment

of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland

dHumboldt-Universität

zu Berlin, Lebenswissenschaftliche Fakultät, Institut für Biologie,

Biophysikalische Chemie, Invalidenstr. 42 10099 Berlin, Germany

*corresponding author e-mail: [email protected] Rifamycins are a group of macrocyclic antibiotics mainly used for the treatment of various bacterial infections including tuberculosis. Spectroscopic studies of rifamycins evidence the formation of temperature- and solvent-dependent equilibria between A-, B-, C-type conformers in solutions. The B- and C-type conformers of rifamycin antibiotics are exclusively formed in the presence of water molecules. A- and B-type conformers exhibit hydrophilic and “open” ansa-bridge nature whereas the C-type conformer is more lipophilic due to the presence of a “closed” ansa-bridge structure. Involvement of the lactam moiety of the ansa-bridge in intramolecular H-bonds within rifapentine and rifampicin implicates formation of a more hydrophilic A-type conformer. In contrast to rifampicin and rifapentine, for rifabutin and rifaximin "free" lactam group enhances conformational flexibility of the ansa-bridge, thereby enabling

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interconversion between A- and C-type conformers. In turn, an equilibrium between A- and C-type conformers for rifamycins improves their adaptation to changing nature of bacteria cell membranes, especially those of Gram-negative strains and/or to efflux pump systems. KEYWORDS:

chameleonic

macrocyclic

drugs,

ansamycins

conformation,

zwitterions, hydrates, membrane permeability, RNA polymerases. The fight against global spread of antibiotic resistance is becoming extremely difficult today due to frequent prescription of these valuable therapeutic agents in not justified cases.1,2 Some bacterial strains are resistant to nearly all known and approved antibiotics, and the design of new ones active against Gram-(+) and Gram-(-) bacterial strains, having different composition of the cell walls, is an important and challenging task.3,4 Gram-(+) bacteria strains, in contrast to Gram-(-) ones, have typically thicker (30-80 nm) and more rigid cell wall built from cytoplasmic lipid membrane (inner), relatively thin periplasmic matrix between inner and outer membranes as well as thick outer peptidoglycan layer composed of: biopolymer Nacetylmuramic acid (NAM), and N-acetylglucosamine (NAG) cross-linked with peptide fragments consisting D-amino acids (D-Ala, D-Glu) and the most hydrophobic and increasing rigidity ingredients called lipoteichoic acids.5 Lipoteichoic acids consist of rybitol and glycerol esterificated by fatty acids having long alkyl tails. In turn, Gram(-) bacteria strain have generally thinner cell envelope than the Gram-(+) one (~2-10 nm), however, of greater complexity i.e. consisting of inner and additionally outer cell membrane.6 The outer cell membrane of Gram-(-) bacteria, lacking of lipoteichoic acids, provides a selective barrier for most molecules of MW < 600-700 thanks to the presence of self-assembled porin units, limiting passage of a more polar molecules. Outer cell membrane of Gram-(-) bacteria contains lipopolysaccharides, whereas the

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periplasmic space, possessing hydrolytic enzymes, is thicker than that of Gram-(+) bacteria. Inner cytoplasmic Gram-(-) bacteria cell membrane, lacking of lipoteichoic acids, is composed of glycerolophospholipids and shows more hydrophobic character. Important problems with designing drugs, able to over-cross especially Gram-(-) bacteria cell walls, are: achieving of expected well-balanced lipophilicity and water solubility parameters, gaining possibility of chameleonic structure changing during transportation of the drug.7–10 Other problem in the selection of leading antibacterials is bacteria resistance reflecting in the presence efflux pump systems and mutations in amino acids sequence of biological targets, as for bacterial RNA polymerases (RNAPs) – molecular target of rifamycin-type antibiotics.11 Semisynthetic

ansa-macrolides

as

rifabutin

(RFB),

rifapentine

(RFP),

rifampicin /also called rifampin/ (RMP) and rifaximin (RFX) (Fig. 1) belong to widely used class of antibiotics.12 Their unique basket-like structure formed by a structural motif called ”ansa-bridge” was extensively studied by NMR spectroscopy in non-polar solutions,13 theoretical14 and X-ray15 methods (Fig. 1). These rifamycin-type antibiotics have identical structures of the ansa-bridge, a comparable aromatic core and relatively strong organic bases at C(3) or C(3)-C(4) arm (Fig. 1). Despite of these common structural features, antibiotics like RFB, RFX and benzoxazinorifamycins functionalized at C(3)-C(4) have high potency against Gram-(+) and Gram-(-) bacterial strains16–19 whereas antibacterial activity of RMP and RFP is mainly restricted to Gram-(+) bacteria and mycobacteria.20,21 Some attempts to improve RMP potency toward Gram-(-) strains by chemo-sensitization using peptidomimetic OAKs (oligo-acyl-lysyls) were earlier undertaken.22 Regardless of whether rifamycin antibiotics are more active toward Gram-(-) or Gram-(+) strains, their general binding mode to the β-subunit of the DNA/RNA channel of bacterial RNAPs is analogous i.e.



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via participation of O(1) and O(8) atoms as well as O(21)H and O(23)H groups in intermolecular interactions with the enzyme.23–26 Hence, it was postulated by Bacchi et al. that rifamycins show reduced biological potency when the O(21)H and O(23)H groups of the ansa-bridge are unavailable for specific intermolecular interactions with the RNAPs due to an orientation of these hydroxyls toward the naphthalene core.15 Our earlier models of the RMP-RNAP complex suggested that the protonated basic C(3) tail of antibiotic is involved in intermolecular H-bond with N448 and E445 from βfork loop 2 of RNAP.27 Taking into account a common binding mode of this type antibiotics to RNAPs (similar docking of the ansa-bridge), the reason of rifamycins different biological potency toward Gram-(+) and Gram-(-) bacteria remains an open question. Floss et al. suggested that the different antibacterial activity of these antibiotics can be linked to transportation of the drug to RNAPs.12 On the contrary, on the basis of sensitivity studies on efflux deficient E. coli (TolC knockout) strain it was concluded that the efflux, but not permeability, is the reason of limited antibacterial potency of rifamycin S, RMP, RFB and RFX.11 In turn, Woodard et al. indicated that E. coli lipopolysaccharide (LPS) deficient strains are more sensitive toward RMP ~500 fold than those without the defect.28 This result shows that RMP, showing concentration-dependent cell killing (e.g. in MTB cells), is easily accumulated in the Gram-negative cells having defective outer polar cell membrane. The explanation of this fact can be attributed to impaired efflux or/and limited adaptation of RMP molecule to changing nature of membranes during transportation. In view of the above informations, the evaluation of structural implications on rifamycins biological mechanism of action at a molecular level is crucial for further rational designing of novel and more potent inhibitors of bacterial RNAPs, both against Gram-(+) and Gram-(-) bacteria strains. For this reason, here we report a new insight into factors


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influencing the ansa-bridge conformation of rifamycin-type antibiotics contributing to better understand structure-activity relationships (SAR).

RESULTS AND DISCUSSION Proton transfer and ansa-bridge arrangement for rifamycin type antibiotics To reveal the structure of the ansa-bridge in different solvents for RMP, RFP, RFX and RFB 1H, 13C and 15N NMR spectra, supported by 2D NMR experiments (1H1H

COSY; 1H-13C HSQC, 1H-15N HSQC, 1H-13C HMBC; 1H-1H NOESY) and FT-IR

spectra were recorded (Figs. 2-3, Tables S1 – S4). The presence of C(8) and C(11) carbon resonances below 170 and above 190 ppm in

13C

NMR spectra, respectively, provides evidence for a non-ionic form of

antibiotics with a C(11)=O ketone group and a phenol at C(8) (Fig. 1, Fig. 2a). In case of a proton transfer from the most acidic phenol O(8)H to the most basic centre within rifamycins, the C(8) and C(11) carbon atom signals appear together in the range 180 - 190 ppm (Fig. 1, Fig. 2a). The close proximity of the C(8) and C(11) carbon atom signals indicates a strong conjugation effect in zwitterion, extending from deprotonated phenol O(8)H to C(11)=O group. Thus, according to

13C

NMR

spectra (Fig. 2a), RMP and RFP exist in solution in: the non-ionic in CDCl3 or zwitterionic form in DMSO-d6+H2O. In turn, RFX exists exclusively in the zwitterionic and RFB exclusively in non-ionic form, irrespective of the solvent used and the presence of water molecules in the solution (Fig. 2a and Tables S2 and S4). In zwitterionic RMP and RFP the proton of the O(8)H phenol is transferred to the basic nitrogen (N40) of the piperazine, as proved by 1H NMR (Fig. 2b) and 1H-15N HSQC studies in solution (Fig. 3a). For RFX the proton transfer process was evidenced by the presence of protonated nitrogen H-N+(4) resonance signal at ~ -330 ppm (1H-15N 5 ACS Paragon Plus Environment


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HSQC in Fig. 3b) and by presence of H-N+(4) proton signal in 1H NMR (Fig. 2b). Due to the repulsion of the negatively charged centers after the hypothetical deprotonation, the presence of analogous zwitterionic form would be unfavorable for RFB (Fig. S1). The non-ionic structure of RFB is confirmed by a chemical shift of the C(8) signal in

13C

NMR spectrum (δC(8)

Specific interactions between rifamycin antibiotics and water

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