Analiza porównawcza własności fizycznych miejsc wiązania antybiotyków aminoglikozydowych w RNA i białkach

Aminoglycoside antibiotics have been in use for more than 60 years, helping combat severe bacterial infections. Due to this long time of usage, more and more bacteria become resistant to one or several drugs from this group. This spread of resistant species is alarming and additionally, there is little knowledge about the mechanisms of bacterial resistance. In order to broaden our understanding of how bacteria combat aminoglycosides, we performed computer simulations of various molecules that bind aminoglycosides in a bacterial cell: (i) the primary binding site, called the A-site and located in ribosomal RNA, wild type and with mutations that decrease the aminoglycoside binding affinity; and (ii) the aminoglycoside modifying enzymes (AMEs), which are produced by bacteria to inactivate these drugs. The mutations of the RNA A-site were chosen based on previous experimental studies on whole bacteria. These studies showed that even single base substitutions were sufficient to make bacteria resistant, but did not explain how this resistance was gained on an atomic level. There are many AMEs and they vary a lot among themselves, yet they all have a narrow specificity towards aminoglycosides, which are quite homogeneous group. The two main questions we have posed in our research are: (i) what are the physical grounds of bacteria becoming less susceptible to aminoglycosides due to RNA A-site mutations; and (ii) how different AMEs attract aminoglycosides and interact with them? We performed all-atom molecular dynamics (MD) simulations of the A-site model with selected mutations and of AME representatives. In addition, the complexes of these biomolecules with aminoglycosides were simulated. For comparison, we also performed simulations of the wild type A-site model and of the aminoglycosides in water. We used various biophysical methods to analyze these simulations and to study: internal dynamics of the biomolecules; electrostatic potential, shape, and volume of the binding pockets; types of interactions with aminoglycosides; and changes in conformations of aminoglycosides. In addition, we developed and implemented an algorithm that helps describe molecular motions. We found that different A-site mutations affect different features of the RNA binding site. Some of them changed the mobility of the nucleic bases, and therefore the shape of the A-site was altered. Other mutations changed the electrostatic potential inside the binding site, thus making it almost unrecognizable to aminoglycosides. The study of AMEs showed that apart from their structural and sequence-related diversity, they differ in the internal movement patterns. However, these enzymes interact with aminoglycosides very similarly, using mainly electrostatic interactions. Interestingly, we noticed that these interactions were copied from the RNA:aminoglycoside complex. Our findings were in agreement with experimental studies and also helped to explain some of their outcomes. The results presented in this dissertation may help design new antibiotics that would overcome the bacterial resistance.Od ponad 60 lat antybiotyki aminoglikozydowe są z powodzeniem stosowane w szpitalach przeciwko ciężkim infekcjom bakteryjnym. Jednak pojawianie się coraz większej liczby przypadków bakterii opornych na stosowane aminoglikozydy sprawia, że badania mechanizmów oporności u bakterii stają kluczowe w dalszej skutecznej walce z infekcjami tego typu. Przeprowadziłam komputerowe symulacje biomolekuł, które oddziałują z antybiotykami aminoglikozydowymi we wnętrzu komórek bakteryjnych. Badanymi obiektami są: (i) główne miejsce wiązania aminoglikozydów, zwane miejscem A, w rybosomalnym RNA; natywne oraz z mutacjami powodującymi wzrost oporności u bakterii; a także (ii) enzymy modyfikujące aminoglikozydy (ang. aminoglycoside modifying enzymes, AME}), produkowane przez bakterie w celu chemicznej dezaktywacji tych leków. Motywacją do badań nad zmutowanym miejscem A był brak informacji o zmianach jakie zachodzą w fizycznych własnościach miejsca A po różnych zamianach nukleotydów. Wiadomo jakie mutacje prowadzą do oporności oraz że nawet pojedyncze zamiany nukleotydu mogą mieć bardzo wymierne skutki, ale nie wyjaśniono jakie są tego podstawy. Natomiast, w przypadku AME, celem prowadzenia symulacji było wyjaśnienie w jaki sposób ta grupa białek jest w stanie być jednocześnie bardzo zróżnicowana i wysoce specyficzna względem aminoglikozydów. Przeprowadziłam symulacje dynamiki molekularnej (MD) modelu miejsca A z wybranymi mutacjami oraz reprezentatywnych enzymów z trzech największych rodzin AME. Aby uzyskać opis oddziaływań między tymi miejscami wiążącymi a aminoglikozydami, przeprowadziłam również symulacje MD tych biomolekuł w kompleksach z wybranymi antybiotykami. W celu analizy symulacji użyłam metodologii z zakresu biofizyki teoretycznej. Badałam wiele własności fizykochemicznych wybranych biomolekuł i ich kompleksów, m.in.: dynamikę wewnętrzną, własności elektrostatyczne, kształt i objętość miejsc wiązania aminoglikozydów, a także rodzaje oddziaływań z aminoglikozydami. Ponadto, stworzyłam nową metodę analizy zmian konformacyjnych w molekułach, która dokonuje podziału biomolekuł na tzw. dynamiczne domeny, na podstawie danych pochodzących z symulacji lub eksperymentów. Z analizy symulacji rybosomalnego miejsca A wynika, że mutacje różnych zasad wpływają na różne własności fizyczne tego fragmentu RNA. W zależności od położenia mutowanej zasady, zmieniał się rozkład ładunków cząstkowych w miejscu wiążącym lub kształt tego miejsca. Mutacje wpływały również na dynamikę ruchów wewnętrznych miejsca A. Analiza symulacji cząsteczek AME wskazała, że oprócz różnorodności struktur trzeciorzędowych i sekwencji, występuje w tej grupie również różnorodność w ruchach wewnętrznych. Pomimo tych różnic, wszystkie enzymy oddziaływały z aminoglikozydami w bardzo podobny sposób, głównie elektrostatycznie. Ponadto, te oddziaływania wydają się być kopiowane z kompleksów, jakie aminoglikozydy tworzą z miejscem A. Rezultaty moich badań są zgodne z poprzednimi doniesieniami eksperymentalnymi, a także pomagają wyjaśnić niektóre z nich. Wyniki opisane w tej pracy mogą być podstawą do zaprojektowania zmodyfikowanych aminoglikozydów, które mogłyby być aktywne nawet wobec opornych bakterii

Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa

Aminoglycoside-3′-Phosphotransferase IIIa is a widespread, promiscuous member of the phosphotransferase family of aminoglycoside modifying enzymes. This study provides results of combined calorimetry/NMR experiments to characterize and dissect the global thermodynamic properties of aminoglycoside–APH(3′)-IIIa complexes. Aminoglycoside binding to APH(3′)-IIIa is enthalpically driven with strong entropic penalty. 2′- and 6′-amino groups have significant contributions to the observed binding parameters. Formation of APH(3′)-IIIa complexes with substrate aminoglycosides shows a complex dependence on pH and is linked to protonation and deprotonation of both ligand and enzyme groups. We report pKa upshifts of ~1 unit for N2′ and N2′′′ groups of enzyme-bound neomycin B while the pKa of N6′ changes by 0.3 unit and N6′′′ experiences no shift. Isotopic solvent and heat capacity change studies strongly suggest differential effects and reorganization of solvent in kanamycin and neomycin class complexes of the enzyme. We also determined unusually high binding ΔCp values in the range of -0.7 to -3.8 kcal/mol·deg which were not explained by changes in the solvent accessible surface area. A break at 30°C was observed in the ΔCp plot and temperaturedependent backbone amide proton chemical shifts of four residues surrounding the binding site of kanamycin-APH(3′)-IIIa complex. These results may indicate specific solvent reorganization sites away from the binding site of the enzyme

Strategies against nonsense: oxadiazoles as translational readthrough-inducing drugs (TRIDs)

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs

Binding thermodynamics of paromomycin, neomycin, neomycin-dinucleotide and -diPNA conjugates to bacterial and human rRNA

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity

Exploring conformational variability of an rna domain in the ribosome: from structure and function to potential antibiotic targeting

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome. Previous model studies using small hairpin H69 showed the conformational differences of H69 loop under different conditions and revealed the significance of modified bases in H69 dynamics. Comparison of crystal structures of ribosomes indicates variable H69 conformations under different conditions. Based on these information, we performed dimethylsulfate (DMS) probing on 50S ribosomal subunits under different pHs, temperatures and Mg2+ concentrations, showing that H69 has a dynamic RNA loop component on the ribosome level as well as the models, and its multiple conformations are dependent on the presence of modified bases. In addition, footprintings in the presence of aminoglycoside antibiotics neomycin and paromomycin also show conformational variability in H69. These results indicate that H69 exists in multiple conformational states, which could be related to the function of the ribosome in the cell

Characterization of a 30S Ribsomal Subunit Intermediate Found in \u3cem\u3eEscherichia coli\u3cem\u3e Cells Growing with Neomycin and Paromomycin.

The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E.coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio labeled antibiotics both in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug in vivo. In addition, cell free reconstitution assays generated a 21S particle during incubation with either aminoglycoside. Precursor formation was inhibited with increasing drug concentration. This work examines features of a novel antibiotic target for aminoglycoside and will provide information that is needed for the design of more effective antimicrobial agents

Doctor of Philosophy

dissertationThe solution structure of domain IIa of the hepatitis C virus internal ribosome entry site in complex with a racemic benzimidazole inhibitor was determined by NMR spectroscopy with corroborating fluorescence data. A 38 base RNA construct representing the inhibitor-binding region of domain IIa was synthesized by T7 RNA polymerase. Fully and selectively 13C and 15N labeled and isotopically unlabeled RNA samples were produced and studied in complex with the inhibitor. The inhibitor was previously shown to have inhibitory activity in an HCV replicon assay. It was also previously found to bind in the bulge region of domain IIa. In the free RNA, this five base bulge region introduces a bend in the extended domain II that situates the terminus of the domain over the mRNA cleft in the ribosomal E site. This hinge-like bulge region is not a known binding site for any host or viral translational cofactors, but domain II has been shown to be critical for IRES function, and the bulgeinduced bend in domain IIa has been shown to be important for IRES function in mutagenesis assays. Molecular dynamics refinement in explicit solvent and subsequent free energetic analysis indicate that the inhibitor enantiomers bind with comparable affinity and equivalent binding modes. The structure of this inhibitor/RNA complex suggests that the small molecule rearranges the base stacking in the bulge and introduces a significant conformational change that eliminates the bent RNA helical trajectory. This suggests a iv possible mechanism of inhibition involving the displacement of the domain II terminus from the mRNA cleft on the ribosomal E site. Perhaps most importantly, this structure may serve as a guide in the development of second-generation higher affinity inhibitors of the hepatitis C IRES as well as provide general insights into small molecule inhibitor interactions with RNA

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)

A study of biomolecular interactions using three biological complexes to explore structure, dynamics and method development

The protein, RDE-4 in C. elegans, served as a model for studying how double stranded RNA binding proteins that bind to dsRNA molecules are critical for RNAi cellular processes. NMR spectroscopy confirms that the RDE-4 construct has characteristic protein domains that bind to. dsRNA and that RNA binding causes a significant global change of the protein structure. SAXS analysis indicates that the two binding domains in the RDE-4 protein do not interact with one another, but instead forms a continuous interface onto which long target dsRNA can bind. Gel shift assay experiments reveal that multiple RDE-4 molecules bind to a non-sequence specific RNA substrate with positive cooperativity. RNA binding occurs with micromolar affinity and a second binding event occurs with millimolar affinity. The binding of E. coli dihydrofolate reductase (DHFR) to inhibitors methotrexate (MTX) and 1,4-Bis-{[ N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (inhibitor 1) has been studied to investigate the dynamics involved in the catalytic mechanism of DHFR. NMR relaxation methods show that in the presence of inhibitor 1, the catalytic domain of DHFR binds 1 in the substrate-binding pocket and an occluded conformation is assumed. In both the DHFR:NADPH and DHFR:NADPH:1 complexes, motion is exhibited on the microsecond-millisecond timescale. The heat capacity change of DHFR upon binding to 1 and MTX are 43 ± 10 cal/mol-K and -120 ± 109 cal/mol-K respectively. Differences in ΔCp of DHFR binding to inhibitor 1 compared to that of MTX indicate that the mode of binding to 1 is different from what is observed in the crystal structure of the complex. An affinity electrophoresis method to screen for RNA-small molecule ligand interactions has been developed. This method is made quantifiable by cross-linking the ligand into the gel matrix and gauging binding by RNA mobility. The utility of this method is demonstrated using the known interaction between different aminoglycoside ligands with the E. coli ribosomal A-site RNA and with an RNA molecule containing a C-C mismatch. Average apparent dissociation constants are determined. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand