Bench to bedside: The ambitious goal of transducing medicinal chemistry from the lab to the clinic.

This paper deals with a critical examination on the possibility of quantitatively predicting the in vivo activity of new chemical entities (NCEs) by making use of in silico and in vitro data including three-dimensional structure of drug-target complex, thermodynamic and crowding parameters, ADME (absorption, distribution, metabolism, excretion) properties, and off-target (toxic) interactions. This formidable challenge is still a dream, given the presently occurring exceedingly high (>95%) attrition rates of NCEs. As a solution we envisage exploiting advanced AI (artificial intelligence) algorithms. In fact, very recent AI implemented programs proved remarkably effective and accurate in predicting the 3D architecture of (any) protein, starting from the amino-acid sequence only. The same accuracy could not be obtained using classical conformational studies. Apart from these breakthrough results, AI algorithms could be profitably used to extract valuable information from the huge amount of data so far accumulated from previous studies. In case of positive results, the drug discovery procedure would be sensibly accelerated, and the relative costs remarkably reduced.

Selective Ligands for Non-Canonical DNA Structures: Do They Have a Future in Medicinal Chemistry?

Winning the war against cancer represents a major goal currently [...].

Targeting Canine KIT Promoter by Candidate DNA G-Quadruplex Ligands.

G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFα, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time- and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.

Highly Improved Electrospray Ionization-Mass Spectrometry Detection of G-Quadruplex-Folded Oligonucleotides and Their Complexes with Small Molecules.

G-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

G-quadruplexes in human promoters: A challenge for therapeutic applications.

BACKGROUND: G-rich sequences undergo unique structural equilibria to form G-quadruplexes (G4) both in vitro and in cell systems. Several pathologies emerged to be directly related to G4 occurrence at defined genomic portions. Additionally, G-rich sequences are significantly represented around transcription start sites (TSS) thus leading to the hypothesis of a gene regulatory function for G4. Thus, the tuning of G4 formation has been proposed as a new powerful tool to regulate gene expression to treat related pathologies. However, up-to date this approach did not provide any new really efficient treatment. SCOPE OF REVIEW: Here, we summarize the most recent advances on the correlation between the structural features of G4 in human promoters and the role these systems physiologically exert. In particular we focus on the effect of G4 localization among cell compartments and along the promoters in correlation with protein interaction networks and epigenetic state. Finally the intrinsic structural features of G4 at promoters are discussed to unveil the contribution of different G4 structural modules in this complex architecture. MAJOR CONCLUSIONS: It emerges that G4s play several roles in the intriguing and complex mechanism of gene expression, being able to produce opposite effects on the same target. This reflects the occurrence of a highly variegate network of several components working simultaneously. GENERAL SIGNIFICANCE: The resulting picture is still fuzzy but some points of strength are definitely emerging, which prompts all of us to strengthen our efforts in view of a selective control of gene expression through G4 modulation. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids.

BACKGROUND: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. METHODS: Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. RESULTS: The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. CONCLUSIONS: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. GENERAL SIGNIFICANCE: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment.

BACKGROUND: G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide. METHODS: Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer. RESULTS: The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context. CONCLUSIONS: CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions. GENERAL SIGNIFICANCE: Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.

Novel ametantrone-amsacrine related hybrids as topoisomerase IIß poisons and cytotoxic agents.

The precise definition of the structural requirements for effective topoisomerase II poisoning by drug molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we prepared several conjugates combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human topoisomerase IIß isoform, and cytotoxic activity resembling that of the parent compounds. In addition, the properties of the lateral groups linked to the anthracenedione group play an important role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19 appear to be promising for further development.

Targeting loop adenines in G-quadruplex by a selective oxirane.

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-ß1 expression in mesangial cells: biochemical and structural considerations.

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-ß1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-ß1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-ß1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-ß1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-ß1 overexpression.

Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates.

Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry.

Quinone methide generation via photoinduced electron transfer.

Photochemical activation of water-soluble 1,8-naphthalimide derivatives (NIs) as alkylating agents has been achieved by irradiation at 310 and 355 nm in aqueous acetonitrile. Reactivity in aqueous and neat acetonitrile has been extensively investigated by laser flash photolysis (LFP) at 355 nm, as well as by steady-state preparative irradiation at 310 nm in the presence of water, amines, thiols, and ethyl vinyl ether. Product distribution analysis revealed fairly efficient benzylation of the amines, hydration reaction, and 2-ethoxychromane generation, in the presence of ethyl vinyl ether, resulting from a [4 + 2] cycloaddition onto a transient quinone methide. Remarkably, we found that the reactivity was dramatically suppressed under the presence of oxygen and radical scavengers, such as thiols, which was usually associated with side product formation. In order to unravel the mechanism responsible for the photoreactivity of these NI-based molecules, a detailed LFP study has been carried out with the aim to characterize the transient species involved. LFP data suggest a photoinduced electron transfer (PET) involving the NI triplet excited state (λ(max) 470 nm) of the NI core and the tethered quinone methide precursor (QMP) generating a radical ions pair NI(•-) (λ(max) 410 nm) and QMP(•+). The latter underwent fast deprotonation to generate a detectable phenoxyl radical (λ(max) 390 and 700 nm), which was efficiently reduced by the radical anion NI(•-), generating detectable QM. The mechanism proposed has been validated through a LFP investigation at 355 nm exploiting an intermolecular reaction between the photo-oxidant N-pentylnaphthalimide (NI-P) and a quaternary ammonium salt of a Mannich base as QMP (2a), in both neat and aqueous acetonitrile. Remarkably, these experiments revealed the generation of the model o-QM (λ(max) 400 nm) as a long living transient mediated by the same reactivity pathway. Negligible QM generation has been observed under the very same conditions by irradiation of the QMP in the absence of the NI. Owing to the NIs redox and recognition properties, these results represent the first step toward new molecular devices capable of both biological target recognition and photoreleasing of QMs as alkylating species, under physiological conditions.

In front of and behind the replication fork: bacterial type IIA topoisomerases.

Topoisomerases are vital enzymes specialized in controlling DNA topology, in particular supercoiling and decatenation, to properly handle nucleic acid packing and cell dynamics. The type IIA enzymes act by cleaving both strands of a double helix and having another strand from the same or another molecule cross the DNA gate before a re-sealing event completes the catalytic cycle. Here, we will consider the two types of IIA prokaryotic topoisomerases, DNA Gyrase and Topoisomerase IV, as crucial regulators of bacterial cell cycle progression. Their synergistic action allows control of chromosome packing and grants occurrence of functional transcription and replication processes. In addition to displaying a fascinating molecular mechanism of action, which transduces chemical energy into mechanical energy by means of large conformational changes, these enzymes represent attractive pharmacological targets for antibacterial chemotherapy.

Effects of magnesium and related divalent metal ions in topoisomerase structure and function.

The catalytic steps through which DNA topoisomerases produce their biological effects and the interference of drug molecules with the enzyme-DNA cleavage complex have been thoroughly investigated both from the biophysical and the biochemical point of view. This provides the basic structural insight on how this family of essential enzymes works in living systems and how their functions can be impaired by natural and synthetic compounds. Besides other factors, the physiological environment is known to affect substantially the biological properties of topoisomerases, a key role being played by metal ion cofactors, especially divalent ions (Mg(2+)), that are crucial to bestow and modulate catalytic activity by exploiting distinctive chemical features such as ionic size, hardness and characteristics of the coordination sphere including coordination number and geometry. Indeed, metal ions mediate fundamental aspects of the topoisomerase-driven transphosphorylation process by affecting the kinetics of the forward and the reverse steps and by modifying the enzyme conformation and flexibility. Of particular interest in type IA and type II enzymes are ionic interactions involving the Toprim fold, a protein domain conserved through evolution that contains a number of acidic residues essential for catalysis. A general two-metal ion mechanism is widely accepted to account for the biophysical and biochemical data thus far available.

Photogeneration and reactivity of naphthoquinone methides as purine selective DNA alkylating agents.

A one-step protecting-group-free synthesis of both 6-hydroxy-naphthalene-2-carbaldehyde and the bifunctional binaphthalenyl derivative afforded 6-hydroxymethylnaphthalen-2-ol, 6-methylaminomethyl-naphthalen-2-ol, [(2-hydroxy-3-naphthyl)methyl]trimethyl ammonium iodide, and a small library of bifunctional binol analogues in good yields. Irradiation of naphthol quaternary ammonium salt and binol-derivatives (X = OH, NHR, NMe(3)(+), OCOCH(3), and L-proline) at 310 and 360 nm resulted in the photogeneration of the 2,6-naphthoquinone-6-methide (NQM) and binol quinone methide analogues (BQMs) by a water-mediated excited-state proton transfer (ESPT). The hydration, the mono- and bis-alkylation reactions of morpholine and 2-ethanethiol, as N and S prototype nucleophiles, by the transient NQM (λ(max) 310, 330 nm) and BQMs (λ(max) 360 nm) were investigated in water by product distribution analysis and laser flash photolysis (LFP). Both the photogeneration and the reactivity of NQM and BQMs exhibited striking differences. BQMs were at least 2 orders of magnitude more reactive than NQM, and they were generated much more efficiently from a greater variety of photoprecursors including the hydroxymethyl, quaternary ammonium salt and several binol-amino acids. On the contrary, the only efficient precursor of NQM was the quaternary ammonium salt. All water-soluble BQM precursors were further investigated for their ability to alkylate and cross-link plasmid DNA and oligonucleotides by gel electrophoresis: the BQMs were more efficient than the isomeric o-BQM (binol quinone methide analogue of 2,3-naphthoquinone-3-methide). Sequence analysis by gel electrophoresis, HPLC, and MS showed that the alkylation occurred at purines, with a preference for guanine. In particular, a BQM was able to alkylate N7 of guanines resulting in depurination at the oligonucleotide level, and ribose loss at the nucleotide level. The photoreactivity of BQM precursors translated into photocytotoxic and cytotoxic effects on two human cancer cell lines: in particular, one compound showed promising selectivity index on both cell lines.

The 6-aminoquinolone WC5 inhibits human cytomegalovirus replication at an early stage by interfering with the transactivating activity of viral immediate-early 2 protein.

WC5 is a 6-aminoquinolone that potently inhibits the replication of human cytomegalovirus (HCMV) but has no activity, or significantly less activity, against other herpesviruses. Here we investigated the nature of its specific anti-HCMV activity. Structure-activity relationship studies on a small series of analogues showed that WC5 possesses the most suitable pattern of substitutions around the quinolone scaffold to give potent and selective anti-HCMV activity. Studies performed to identify the possible target of WC5 indicated that it prevents viral DNA synthesis but does not significantly affect DNA polymerase activity. In yield reduction experiments with different multiplicities of infection, the anti-HCMV activity of WC5 appeared to be highly dependent on the viral inoculum, suggesting that WC5 may act at an initial stage of virus replication. Consistently, time-of-addition and time-of-removal studies demonstrated that WC5 affects a phase of the HCMV replicative cycle that precedes viral DNA synthesis. Experiments to monitor the effects of the compound on virus attachment and entry showed that it does not inhibit either process. Evaluation of viral mRNA and protein expression revealed that WC5 targets an event of the HCMV replicative cycle that follows the transcription and translation of immediate-early genes and precedes those of early and late genes. In cell-based assays to test the effects of WC5 on the transactivating activity of the HCMV immediate-early 2 (IE2) protein, WC5 markedly interfered with IE2-mediated transactivation of viral early promoters. Finally, WC5 combined with ganciclovir in checkerboard experiments exhibited highly synergistic activity. These findings suggest that WC5 deserves further investigation as a candidate anti-HCMV drug with a novel mechanism of action.

Simocyclinone D8 turns on against Gram-negative bacteria in a clinical setting.

Simocyclinone D8 (SD8) is known to affect Gram-positive bacteria only. By testing SD8 against several clinical isolates, we showed that SD8 resulted very active against Gram-negative bacteria from clinical specimens, while it was shown inactive against laboratory strains. The activity against the former was in part due to enhanced drug entry. In addition, SD8 appears to share chromosome- and plasmid-mediated resistance mechanisms with fluoroquinolones.

Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

Quinone methides tethered to naphthalene diimides as selective G-quadruplex alkylating agents.

We have developed novel G-quadruplex (G-4) ligand/alkylating hybrid structures, tethering the naphthalene diimide moiety to quaternary ammonium salts of Mannich bases, as quinone-methide precursors, activatable by mild thermal digestion (40 degrees C). The bis-substituted naphthalene diimides were efficiently synthesized, and their reactivity as activatable bis-alkylating agents was investigated in the presence of thiols and amines in aqueous buffered solutions. The electrophilic intermediate, quinone-methide, involved in the alkylation process was trapped, in the presence of ethyl vinyl ether, in a hetero Diels-Alder [4 + 2] cycloaddition reaction, yielding a substituted 2-ethoxychroman. The DNA recognition and alkylation properties of these new derivatives were investigated by gel electrophoresis, circular dichroism, and enzymatic assays. The alkylation process occurred preferentially on the G-4 structure in comparison to other DNA conformations. By dissecting reversible recognition and alkylation events, we found that the reversible process is a prerequisite to DNA alkylation, which in turn reinforces the G-quadruplex structural rearrangement.