MOLECULAR MODELLING STUDIES OF THE NF-κB BIOLOGICAL SYSTEM AS RELEVANT DRUG TARGET

Background Virtual screening (VS) is now a well-established method for finding small molecular modulators of a biological relevant macromolecule function by using much older computational techniques, such as docking simulations. The NF-kB family consists of a group of eukaryotic inducible transcription factors that have evoked widespread interest until now. As a matter of fact, the dysfunction of NF-kB is associated with many serious disease states. In spite of the well-defined solved NF-kB 3D structures, to the best of our knowledge, nowadays VS applications against this interesting target for the discovery of new NF-kB/DNA inhibitors have not been published yet. Aims The project described in this thesis is aimed to the identification of low molecular weight compounds interacting with NF-kB and able to inhibit DNA/NF-kB complex formation and biological dependent functions. Methods As a first example towards the development of a docking protocol fit to the target under study (NF-kB p50 monomer and homodimer), a data set of 27 bioactive natural compounds were prepared. In the second example a chemical library was purpose-made for VS applying a filtering procedure on 3D ZINC commercial database containing more than 2 million compounds. Successively a focus furocoumarin library of 1700 molecules was prepared for subsequent docking runs. The three dimensional structure of the complex NF-kB-DNA was retrieved from the RCSB Protein Data Bank. The cocrystallized DNA macromolecule was removed from the structure. p50-p50 homodimer, p50-p65 heteodimer and p50 monomers were selected for the docking simulations and prepared using the graphical interface Maestro. All molecules under study were docked in to the binding site of the target using Glide software from Schrodinger. Grids were prepared for each proteins. The coordinates of the enclosing box were defined starting from the set of active site residues involved in hydrogen bonds with the NF-kB recognition site of DNA and optimised including the double strands DNA helices volume by visual inspection. Docking studies were based on rigid X-ray crystal transcription factor structures (1LE9, 1IKN) and no information is reported in literature on dynamic features of this protein. Thus, 14ns of canonical MD simulations were carried out in explicit solvent on structural models of NF-kB systems as free and bound both to DNA and IkB. CHARMM27 force field, TIP3P model for water and P.M.E. for electrostatic force calculation were used. Main Results We found that the adopted docking sampling method and the scoring function were successful in predict the ranking of active and inactive natural compounds into DNA binding site of the ensemble protein (NF-kB p50). Further EMSA assays and biological evaluation were in agreement with computational findings. A successful identification of a micromolar active compound from docking-based VS of the prepared compound library is described. Subsequent in silico screening of the developed focus furocoumarin library carried forward the identifications of five micromolar p50 binders. The binding modes of the best-scored compounds showed the involvement of an essential residue for p50 in vivo activity in DNA interaction. In depth investigations of these interesting hits are also reported, including docking analysis into p50-p65 heterodimer. The ligands showed a different occupation of the DNA binding region of the heterodimer. The structural and dynamics properties of NF-kB p50-p65 in free form and in complex with DNA and IkB have been analyzed showing quite pertinent differences. The most relevant configurations of the transcription factor have been clustered and selected for further docking studies and drug design. Conclusions Discovered micromolar bioactive compounds can be considered as “leads” for developing stronger NF-kB/DNA inhibitors. The encouraging computational and biological results here reported could open new perspectives in discovery of NF-kB/DNA interaction inhibitors by taking advantages from recent VS strategies

Docking of molecules identified in bioactive medicinal plants extracts into the p50 NF-kappaB transcription factor: correlation with inhibition of NF-kappaB/DNA interactions and inhibitory effects on IL-8 gene expression

<p>Abstract</p> <p>Background</p> <p>The transcription factor NF-kappaB is a very interesting target molecule for the design on anti-tumor, anti-inflammatory and pro-apoptotic drugs. However, the application of the widely-used molecular docking computational method for the virtual screening of chemical libraries on NF-kappaB is not yet reported in literature. Docking studies on a dataset of 27 molecules from extracts of two different medicinal plants to NF-kappaB-p50 were performed with the purpose of developing a docking protocol fit for the target under study.</p> <p>Results</p> <p>We enhanced the simple docking procedure by means of a sort of combined target- and ligand-based drug design approach. Advantages of this combination strategy, based on a similarity parameter for the identification of weak binding chemical entities, are illustrated in this work with the discovery of a new lead compound for NF-kappaB. Further biochemical analyses based on EMSA were performed and biological effects were tested on the compound exhibiting the best docking score. All experimental analysis were in fairly good agreement with molecular modeling findings.</p> <p>Conclusion</p> <p>The results obtained sustain the concept that the docking performance is predictive of a biochemical activity. In this respect, this paper represents the first example of successfully individuation through molecular docking simulations of a promising lead compound for the inhibition of NF-kappaB-p50 biological activity and modulation of the expression of the NF-kB regulated IL8 gene.</p

Synthesis and biological evaluation of 2-amino-3-(3 ',4 ',5 '-trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents

A new series of compds. in which the 2-amino-5-chlorophenyl ring of 3,4,5-(MeO)3C6H2COC6H4(NH2)Cl-2,5 was replaced with a 2-amino-5-arylthiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymn. and colchicine binding to tubulin. 2-Amino-3-(3,4,5-trimethoxybenzoyl)-5-phenylthiophene and its 4-F (I), 4-Me (II), and 4-MeO derivs. displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. I and II were more active than combretastatin A-4 as inhibitors of tubulin polymn. Mol. docking simulations to the colchicine site of tubulin were performed to det. the possible binding mode of I. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymn. and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compds

Identification of an achiral analogue of J-113397 as potent nociceptin/orphanin FQ receptor antagonist

To date, J-113397 represents the most potent and selective non peptide NOP receptor antagonist widely used in pharmacological studies. However, the synthesis, purification, and enantiomer separation of this molecule, which contains two chiral centers, is rather difficult and low-yielding. Here, we synthesized and tested a series of simplified J-113397 analogues to investigate the importance of the stereochemistry and the influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen atom of the benzimidazolidinone nucleus. The compound coded as Trap-101, an achiral analogue of J-113397, combines a pharmacological profile similar to that of the parent compound with a practical, high-yielding preparation

Novel 1,3-Dipropyl-8-(3-benzimidazol-2-yl-methoxy-1-methylpyrazol-5-yl)xanthines as Potent and Selective A_2B Adenosine Receptor Antagonists

Molecular modeling studies, including the comparative molecular field analysis (CoMFA) method, on 52 antagonists of the A_2B adenosine receptor with known biological activity were performed to identify the three-dimensional features responsible for A_2B adenosine receptor antagonist activity. On the basis of these and previous results on the potent antagonist effect of 8-pyrazolyl-xanthines at human A_2BAR, a new series of compounds was synthesized and evaluated in binding studies against the human A₁, A_2A, A₃, and A_2BARs. A remarkable improvement in selectivity with respect to the previous series, maintaining the potency at human A_2B receptor, was achieved, as exemplified by the 8-[3-(4-chloro-6-trifluoromethyl-1<i>H</i>-benzoimidazol-2-yl-methoxy)-1-methyl-1<i>H</i>-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione derivative <b>66</b>: <i>K</i>_i A_2B = 9.4 nM, IC₅₀ hA_2B = 26 nM hA₁/hA_2B = 269, hA_2A/hA_2B > 106, hA₃/hA_2B >106. This study also led to the identification of a series of pyrazole-xanthine compounds with a simplified structure, exemplified by 8-(3-hydroxy-1-methyl-1<i>H</i>-pyrazol-5-yl)-xanthine <b>80</b> displaying very high affinity at A_2BAR with good selectivity over AR subtypes (<i>K</i>_i = 4.0 nM, IC₅₀ hA_2B = 20 nM hA₁/hA_2B = 183, hA_2A,hA₃/hA_2B > 250)