Conformational Equilibria Landscapes: Rotational Spectroscopy and Modeling of Isolated Molecular Systems

To design drugs, molecular designers need exhaustive information on the energetic and structural factors that drive the conformational preferences at both free and bound states. These information can be inferred from structural analysis of molecule or model molecular systems. Rotational spectroscopy analysis combined to theoretical methods provide synergic approachs to investigate, in detail, the structure and internal dynamics of both isolated molecules and weakly bound complexes. In free-jet rotational spectroscopy, the experimental measurements are done by microwave spectrometers operating in the unperturbed environment of a jet plume. While the interpretation of the experimental data is performed by theoretical methods that use semi rigid and coupled Hamiltonians, ab initio and DFT calculations and flexible models. The rotational spectra are highly sensitive to the atomic masses distribution, so conformational equilibria and isotopologues species can be investigated. Regarding internal dynamics, useful information, are obtained, when hyperfine structures, are observed in the spectra. Owed to these information, it is possible to model the potential energy surface governing these motions. Furthermore, millimeter wave spectroscopy contributes significantly to the astrochemistry research area. A lot of information on circumstellar and interstellar medium results from the study of the electromagnetic radiation that reaches us. High resolution and sensitive radio-astronomy tools such as the telescopes Atacama Large Millimeter/submillimeter Array (ALMA), Herschel Space Observatory for the Far-Infrared, have been built to pick up these radiations. Laboratory rotational spectroscopy provides the basic physical parameters necessary for interpreting the astronomical spectra. During my PhD, several molecules and weakly bound complexes have been characterized using the rotational spectroscopy technique and theoretical calculations. The most of my work has been performed in the laboratory of rotational spectroscopy at UNIBO, while some objectives have been analyzed in the course of my visit at laboratory of prof. Sanz in King’s College Department in London

Structure, Dynamics, and Accurate Laboratory Rotational Frequencies of the Acrylonitrile–Methanol Complex

The hydrogen-bonded complex between acrylonitrile (CH2=CHCN) and methanol has been characterized spectroscopically in the millimeter wave range (59.6-74.4 GHz) using a free jet absorption millimeter wave spectrometer. Precise values of the rotational and centrifugal distortion constants were obtained from the measured frequencies of the complex of acrylonitrile with CH3OH and CD3OD. The analysis of the splittings of the rotational lines due to the hindered internal rotation of the methanol methyl group led to the determination of a V-3 value of 221.9(7) and 218(5) cm(-1) for the complexes of CH3OH and CD3OD, respectively, and these values are about 40% lower than that of free methanol. The structure of the observed conformation is in agreement with the global minimum determined at the MP2/aug-cc-pVTZ level of calculation, and the counterpoise corrected intermolecular binding energy, obtained at the same theoretical level, is D-e = 26.3 kJ mol(-1)We thank the University of Bologna for funding (RFO). C.C. acknowledges the Spanish Government (MINECO, Project Code CTQ2017-89150-R) for a postdoctoral contract. W.D.G. acknowledges support from the Swedish Research Council (Vetenskapsradet), Grant Number 2019-04332. We thank R. A. Boto and J. Contreras-Garcia for the availability of the NCI 2D plot script

Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-κB pathway

The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-κB (NF-κB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-κB binding sites in the miR-155 promoter; both sites recruited NF-κB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-κB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-κB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations

Molecular docking of analogues linezolid

Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It appeared on the market in 2000 for treatment of infections caused by streptococci, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus. Linezolid acts as a protein synthesis inhibitor by binding to the ribosomal peptidyl transferase center and stopping the growth of bacteria [1]. In vitro and in vivo assays of oxazolidinone analogues show that the structural features to have a good biological activity and pharmacokinetic properties are: 1) an N-aryl group, 2) a (5S)-absolute configuration, 3) the preference for a C-5 acetamidomethyl side chain, 4) unsaturated or electron-withdrawing groups in the para position of the N-aryl ring generally potentiate activity but an electron-donating amino substituent in the para position on the phenyl ring can confer a good safety profile, 5) additional substitutions on the proximal aromatic ring or a C-4 or C-5 of the oxazolidinone ring usually a detrimental or at best, indifferent effect on the antibacterial activity, 6) one or two fluorine atoms on the phenyl ring usually exerts a significant potentiating effect on efficacy [2]. However the rising resistance to linezolid of some bacterial strains necessitates the discovery of novel biological active molecules. In this study we elucidate the binding mode of linezolid and its analogues in the apo and holo ribosomal subunit of Haloarcula Marismortui bacterium with the molecular docking software AutoDock 4.2 [3]. The ligands chosen are three commercial oxazolidinone compounds (posizolid, eperezolid, torezolid) in which morpholino ring and acetamidomethyl residue are replaced and four new-synthesized molecules in which the oxazolidinone ring is replaced. Early work in this area identified suitable bioisosteric replacement for the usual oxazolidinone ring was: 1) a 5-membered ring appears optimal, 2) a sp2 center adjacent to the phenyl ring, 3) an oxygen atom strategically bound, 4) a chiral center of appropriate absolute configuration. Since these above principles, the isoxazolidinone and dihydropyrrole rings have been chosen like suitable ring replacements [2]. The ligands have on C-4 position ring isopropyl and cyclohesyl substituents. These ligands have been synthesized for the first time in the Alessandra Tolomelli's laboratory (Department of Chemistry "G. Ciamician" University of Bologna, Italy). Docked posizolid molecule in the holo ribosomal binding site. The 5-membered ring orients toward the ribosomal tunnel whereas 2,3 dihydroxyacetone is toward the intersubunit interface. Within the pocket the oxazolidonone ring is stacked against the base moiety of U2539. The dihydroxyacetone OH groups participate in hydrogen bonds with the ribose group of U2541 and U2619. The fluorophenyl moiety makes stacking interaction with the residues A2486 and C2487, whereas the tetrahydropyridine ring does not appear to make significant interactions with the ribosome. The obtained results for each ligand are presented and discussed. The predictions show good reliability for the holo receptor but produce substantially different results for apo one. This is probably due to the complexity of the receptor, formed by proteins and nucleic acids. We are thus improving the docking scheme by including some degrees of flexibility in the receptor and by testing different docking algorithms. These tasks are currently being addressed in our laboratories. [1] J.A. Ippolito, Z. Kanyo, D. Wang, F. Franceschi, P. Moore, T. Steitz, and E. Duffy, Journal medical chemistry, 2008, 51, 3353-3356. [2] Antibiotic discovery and development. T.J. Dougherty, and M.J. Pucci, Michael, Springer ed., 2012, XVll, 273-293. [3] Journal of computational chemistry R. Huey, G. Morris, and A. Olson, D. Goodsell, 2007, 28, 1145-1152

Rotational spectroscopy of non-covalently bound complexes of medium size organic molecules

The conformational space of non-covalently bound complexes of medium size organic molecules is shaped by competing interactions occurring within the molecules or with the partners. It usually presents a high number of low energy conformations very close in energy with shallow potential energy barriers through which the molecular system can tunnel. The conformational preferences of non-covalently bound complexes can be studied to a very high degree of accuracy by free jet rotational spectroscopy1 and from the detailed structural and dynamical data that can be obtained, the site and geometry of the interaction and information on the binding energy can be inferred without ambiguity. The questions usually addressed are: which is the preferred binding site, which type of interactions are established, and whether any conformational change takes place in the monomers upon complexation. Answers to these questions allow insight into the molecular interaction process at the molecular level, bridging the gap between gas-phase and bulk properties. Chosen examples of published and unpublished results of complexes of medium-size organic molecules with different partners formed in a supersonic expansion and characterized by rotational spectroscopy will be discussed. The partner molecules are held together by hydrogen bonds, weak hydrogen bonds and lone-pair-\uf070-hole interactions. It will be shown how non-bonding interactions compete to shape the conformational space of the complexes, the structural changes brought to the conformers of the monomers by complexation and how these interactions can be drastically changed through atomic or functional group substitution

High Resolution Free Jet Millimeter Wave Absorption Spectroscopy: a bridge to Astrochemistry

Conventional absorption spectroscopy is still the workhorse in high-resolution rotational laboratory spectroscopy.1 The data obtained from these kind of instruments are relevant for astronomical searches of complex molecules that represent excellent probes of the physical and chemical environments and history of the sources where they are detected.2 Nowadays, observations performed by the Atacama Large Millimeter Array (ALMA) open up new opportunities to reveal the chemical complexity of solar systems analogues. At the same time the huge amount of data collected and the extremely rich surveys represent a challenge for the astrochemistry community. The chance to detect molecules with an increasing large number of atoms, goes hand in hand with the complexity of their conformational equilibria, often associated with large amplitude motions, that need to be analysed in laboratory before taking on an astronomical search. For this reason a strong interplay between the laboratory spectroscopists and the observational astronomers is increasingly required to be able to unravel the spectra, which are rather difficult to predict theoretically, mainly in the sub-mm wave region. In this talk laboratory results on diols and thiols of potential astronomical interest, obtained using the only Free Jet Absorption Millimeter Wave (FJAMMW) spectrometer working at the University of Bologna,3 will be presented. The rotational spectra (59.6 - 74.4 GHz, corresponding to 5.03 - 4.03 mm) reveal the presence of six conformers for 1,2-butanediol (C4H10O2) and four conformers for 1,3-propanedithiol (C3H8S2), proving the complexity of the conformational landscapes of these kind of compounds. Moreover, taking advantage of the existing public ALMA data, some considerations on the rich molecular line spectrum of the Class 0 protostellar binary IRAS 16293-2422 will be discussed. References [1] S. Br\ufcnken, S. Schlemmer, arXiv:1605.07456, 2016 [2] E. Herbst, E. F. van Dishoeck, Annu. Rev. Astron. Astrophys. 47, 427, 2009 [3] C. Calabrese, A. Maris, L. Evangelisti, L. B. Favero, S. Melandri, W. Caminati, J. Phys. Chem. A. 117, 13712, 201

On the torsional flexibility of the dihydrolipoic acid's pharmacophore: 1,3-propanedithiol

The conformational space of the antioxidant dihydrolipoic acid has been explored through the investigation of its pharmacophore 1,3-propanedithiol. Five of the possible 25 non equivalent isomers (namely: gGGg\u2019, gGGg, g\u2019AGg, gAGg and g\u2019AGg\u2019) were observed in the 59.6-74.4 GHz frequency region by free-jet absorption rotational spectroscopy and for three of them also the 34S mono-substituted isotopologues were detected in natural abundance. Theoretical simulations show that the balance of steric and electronic intramolecular interactions arises on a shallow conformational potential energy surface and suggest that in polar solvents the dithiol chain\u2019s flexibility is greater than in the isolated phase