Quadruplex-duplex junction in LTR-III: A molecular insight into the complexes with BMH-21, namitecan and doxorubicin.

Quadruplex-Duplex (Q-D) junctions are unique structural motifs garnering increasing interest as drug targets, due to their frequent occurrence in genomic sequences. The viral HIV LTR-III sequence was chosen as a Q-D junction model to study the affinity of the selected compounds BMH-21, namitecan (ST-1968), and doxorubicin (DOXO), all containing a planar polycyclic aromatic moiety, linked to either one short aminoalkyl or an aminoglycosyl group. A multidisciplinary approach that combines NMR spectroscopy, molecular modelling, circular dichroism (CD) and fluorescence spectroscopy was employed. The studied ligands induced moderate but clear stabilization to the Q-D junction by interacting with the interfacial tetrad. DOXO was found to be the best Q-D junction binder. Interestingly, the removal of the aminoglycosyl group significantly changed the pattern of the interactions, indicating that highly polar substituents have a stronger affinity with the exposed regions of the Q-D junction, particularly at the level of the interfacial tetrad.

Exploring the Interaction of New Pyridoquinazoline Derivatives with G-Quadruplex in the c-MYC Promoter Region.

Novel amino-substituted pyridoquinazolinone derivatives have been designed and synthesized as potential c-MYC G-quadruplex (G4) ligands, employing an efficient methodology. All the new compounds exhibited moderate to good antiproliferative activity against the human osteosarcoma U2OS cell line. NMR and docking experiments revealed that the recently synthesized compounds interact with the Pu22 G-quadruplex in the c-MYC promoter region, establishing a 2:1 complex, with each molecule positioned over the tetrads at the 3'- and 5'-ends.

Design and Pharmacological Characterization of α4ß1 Integrin Cyclopeptide Agonists: Computational Investigation of Ligand Determinants for Agonism versus Antagonism.

α4ß1 integrin is a cell adhesion receptor deeply involved in the migration and accumulation of leukocytes. Therefore, integrin antagonists that inhibit leukocytes recruitment are currently regarded as a therapeutic opportunity for the treatment of inflammatory disorder, including leukocyte-related autoimmune diseases. Recently, it has been suggested that integrin agonists capable to prevent the release of adherent leukocytes might serve as therapeutic agents as well. However, very few α4ß1 integrin agonists have been discovered so far, thus precluding the investigation of their potential therapeutic efficacy. In this perspective, we synthesized cyclopeptides containing the LDV recognition motif found in the native ligand fibronectin. This approach led to the discovery of potent agonists capable to increase the adhesion of α4 integrin-expressing cells. Conformational and quantum mechanics computations predicted distinct ligand-receptor interactions for antagonists or agonists, plausibly referable to receptor inhibition or activation.

Investigation of the Interaction between Aloe vera Anthraquinone Metabolites and c-Myc and C-Kit G-Quadruplex DNA Structures

G-quadruplexes are nucleotide sequences present in the promoter region of numerous oncogenes, having a key role in the suppression of gene transcription. Recently, the binding of anthraquinones from Aloe vera to G-quadruplex structures has been studied through various physico-chemical techniques. Intrigued by the reported results, we investigated the affinity of aloe emodin, aloe emodin-8-glucoside, and aloin to selected G-quadruplex nucleotide sequences by NMR spectroscopy. The structural determinants for the formation of the ligand/nucleotide complexes were elucidated and a model of the interactions between the tested compounds and C-Kit and c-Myc G-quadruplex DNA structures was built by integrated NMR and molecular modeling studies. Overall, the obtained results confirmed and implemented the previously reported findings, pointing out the complementarity of the different approaches and their contribution to a more detailed overview of the ligand/nucleotide complex formation. Furthermore, the proposed models of interaction could pave the way to the design of new nature-derived compounds endowed with increased G-quadruplex stabilizing activity.

Exploring the Interaction of G-quadruplex Binders with a (3 + 1) Hybrid G-quadruplex Forming Sequence within the PARP1 Gene Promoter Region.

The enzyme PARP1 is an attractive target for cancer therapy, as it is involved in DNA repair processes. Several PARP1 inhibitors have been approved for clinical treatments. However, the rapid outbreak of resistance is seriously threatening the efficacy of these compounds, and alternative strategies are required to selectively regulate PARP1 activity. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter was recently identified. In this study, we explore the interaction of known G-quadruplex binders with the G-quadruplex structure found in the PARP gene promoter region. The results obtained by NMR, CD, and fluorescence titration, also confirmed by molecular modeling studies, demonstrate a variety of different binding modes with small stabilization of the G-quadruplex sequence located at the PARP1 promoter. Surprisingly, only pyridostatin produces a strong stabilization of the G-quadruplex-forming sequence. This evidence makes the identification of a proper (3+1) stabilizing ligand a challenging goal for further investigation.

Alkaloid Escholidine and Its Interaction with DNA Structures.

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers.

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.

G-quadruplex binding properties of a potent PARP-1 inhibitor derived from 7-azaindole-1-carboxamide.

Poly ADP-ribose polymerases (PARP) are key proteins involved in DNA repair, maintenance as well as regulation of programmed cell death. For this reason they are important therapeutic targets for cancer treatment. Recent studies have revealed a close interplay between PARP1 recruitment and G-quadruplex stabilization, showing that PARP enzymes are activated upon treatment with a G4 ligand. In this work the DNA binding properties of a PARP-1 inhibitor derived from 7-azaindole-1-carboxamide, (2-[6-(4-pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-b]pyridin-1-yl]-acetamide, compound 1) with model duplex and quadruplex DNA oligomers were studied by NMR, CD, fluorescence and molecular modelling. We provide evidence that compound 1 is a strong G-quadruplex binder. In addition we provide molecular details of the interaction of compound 1 with two model G-quadruplex structures: the single repeat of human telomeres, d(TTAGGGT)4, and the c-MYC promoter Pu22 sequence. The formation of defined and strong complexes with G-quadruplex models suggests a dual G4 stabilization/PARP inhibition mechanism of action for compound 1 and provides the molecular bases of its therapeutic potential.

Pharmacological Characterization of µ-Opioid Receptor Agonists with Biased G Protein or ß-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation.

In recent years, G protein vs. ß-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the ß-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure ß-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand-receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or ß-arrestin.

Putative SARS-CoV-2 Mpro Inhibitors from an In-House Library of Natural and Nature-Inspired Products: A Virtual Screening and Molecular Docking Study.

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) has been the cause of a recent global pandemic. The highly contagious nature of this life-threatening virus makes it imperative to find therapies to counteract its diffusion. The main protease (Mpro) of SARS-CoV-2 is a promising drug target due to its indispensable role in viral replication inside the host. Using a combined two-steps approach of virtual screening and molecular docking techniques, we have screened an in-house collection of small molecules, mainly composed of natural and nature-inspired compounds. The molecules were selected with high structural diversity to cover a wide range of chemical space into the enzyme pockets. Virtual screening experiments were performed using the blind docking mode of the AutoDock Vina software. Virtual screening allowed the selection of structurally heterogeneous compounds capable of interacting effectively with the enzymatic site of SARS-CoV-2 Mpro. The compounds showing the best interaction with the protein were re-scored by molecular docking as implemented in AutoDock, while the stability of the complexes was tested by molecular dynamics. The most promising candidates revealed a good ability to fit into the protein binding pocket and to reach the catalytic dyad. There is a high probability that at least one of the selected scaffolds could be promising for further research.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

Endomorphin-2 analogs containing modified tyrosines: Biological and theoretical investigation of the influence on conformation and pharmacological profile.

New analogs of the endogenous opioid agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) have been obtained by introducing modified tyrosines at the position 1 of the sequence. For all analogs, the cis/trans conformation ratio about the tyramine-Pro amide bond, lipophilicity, receptor affinities, and functional activities, have been determined. Among the novel derivatives, [Dmt(3'-Cl)]1EM-2 (4) stood out for its subnanomolar µ-opioid receptor affinity and potent agonist activity, superior to that of the parent peptide EM-2. Hybrid quantum mechanics/molecular mechanics docking computations supported the cis tyramine-Pro bioactive conformation, and allowed us to analyze the contribution of the substituents of the "message" tyramine to binding, highlighting the role of halogen-bonding in the higher receptor affinity of peptide 4.

Hybrid topoisomerase I and HDAC inhibitors as dual action anticancer agents.

Recent studies have shown that HDAC inhibitors act synergistically with camptothecin derivatives in combination therapies. To exploit this synergy, new hybrid molecules targeting simultaneously topoisomerase I and HDAC were designed. In particular, a selected multivalent agent containing a camptothecin and a SAHA-like template showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Constraining Endomorphin-1 by ß,α-Hybrid Dipeptide/Heterocycle Scaffolds: Identification of a Novel κ-Opioid Receptor Selective Partial Agonist.

Herein we present the expedient synthesis of endomorphin-1 analogues containing stereoisomeric ß2-homo-Freidinger lactam-like scaffolds ([Amo2]EM), and we discuss opioid receptor (OR) affinity, enzymatic stability, functional activity, in vivo antinociceptive effects, and conformational and molecular docking analysis. Hence, H-Tyr-Amo-Trp-PheNH2 resulted to be a new chemotype of highly stable, selective, partial KOR agonist inducing analgesia, therefore displaying great potential interest as a painkiller possibly with reduced harmful side effects.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

BACKGROUND: Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. METHODS: The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. RESULTS AND CONCLUSIONS: We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. GENERAL SIGNIFICANCE: Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis.

Camptothecin-psammaplin A hybrids as topoisomerase I and HDAC dual-action inhibitors.

Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of camptothecin derivatives and HDAC inhibitors. To exploit this synergy in a single active compound, we designed new dual-acting multivalent molecules simultaneously targeting topoisomerase I and HDAC. In particular, a selected compound containing a camptothecin and the psammaplin A scaffold showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016.

Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22.

BACKGROUND: Intra-molecular G-quadruplex structures are present in the guanine rich regions of human telomeres and were found to be prevalent in gene promoters. More recently, the targeting of c-MYC transcriptional control has been suggested, because the over expression of the c-MYC oncogene is one of the most common aberration found in a wide range of human tumors. METHODS: The interaction of nemorubicin and doxorubicin with DNA G-quadruplex structures has been studied by NMR, ESI-MS and molecular modelling, in order to obtain further information about the complex and the multiple mechanisms of action of these drugs. RESULTS AND CONCLUSIONS: Nemorubicin intercalates between A3 and G4 of d(TTAGGGT)4 and form cap-complex at the G6pT7 site. The presence of the adenine in this sequence is important for the stabilization of the complex, as was shown by the interaction with d(TTGGGTT)4 and d(TTTGGGT)4, which form only a 1:1 complex. The interaction of doxorubicin with d(TTAGGGT)4 is similar, but the complex appears less stable. Nemorubicin also binds with high efficiency the c-MYC G-quadruplex sequence Pu22, to form a very well defined complex. Two nemorubicin molecules bind to the 3'-end and to the 5'-end, forming an additional plane of stacking over each external G-tetrad. The wild type c-MYCPu22 sequence forms with nemorubicin the same complex. GENERAL SIGNIFICANCE: Nemorubicin and doxorubicin, not only intercalate into the duplex DNA, but also result in significant ligands for G-quadruplex DNA segments, stabilizing their structure; this may in part explain the multiple mechanisms of action of their antitumor activity.

Synthesis of mixed MOR/KOR efficacy cyclic opioid peptide analogs with antinociceptive activity after systemic administration.

Cyclic pentapeptide Tyr-c[D-Lys-Phe-Phe-Asp]NH2, based on the structure of endomorphin-2 (EM-2), which shows high affinity to the µ-opioid receptor (MOR) and a very strong antinociceptive activity in mice was used as a parent compound for the structure-activity relationship studies. In this report we synthesized analogs of a general sequence Dmt-c[D-Lys-Xaa-Yaa-Asp]NH2, with D-1- or D-2-naphthyl-3-alanine (D-1-Nal or D-2-Nal) in positions 3 or 4. In our earlier papers we have indicated that replacing a phenylalanine residue by the more extended aromatic system of naphthylalanines may result in increased bioactivities of linear analogs. The data obtained here showed that only cyclopeptides modified in position 4 retained the sub-nanomolar MOR and nanomolar κ-opioid receptor (KOR) affinity, similar but not better than that of a parent cyclopeptide. In the in vivo mouse hot-plate test, the most potent analog, Dmt-c[D-Lys-Phe-D-1-Nal-Asp]NH2, exhibited higher than EM-2 but slightly lower than the cyclic parent peptide antinociceptive activity after peripheral (ip) and also central administration (icv). Conformational analyses in a biomimetic environment and molecular docking studies disclosed the structural determinants responsible for the different pharmacological profiles of position 3- versus position 4-modified analogs.