1. Inhibition of Hedgehog Pathway
We possess a unique library of bioactive natural compounds (up to 1000), for in vitro and in vivo activity screenings. All compounds have been clustered based on their structural similarities. We focused our attention on the Hedgehog (Hh) signalling pathway which plays a pivotal role in the initiation, proliferation, invasion and metastasis of various cancers. Smoothened Receptor (Smo) is the most widely appreciated drug target of the Hedgehog signalling pathway. Compounds under clinical investigations are all Smo antagonists. However, medulloblastoma (MB) and other Hh-dependent tumors harbour Smo-indipendent increased function of the downstream Glioma-associated oncogene homolog 1 (or Gli1) effectors, due to high protein levels or activatory mechanisms, raising the need to identify novel drugs blocking Hedgehog pathway downstream of Smoothened. The redundancy of such Gli-activating mechanisms may represent a challenge for the design of targeted personalized therapies.
Based on the knowledge of the crystallographic structure of the zinc finger domain of Gli1 (Gli1ZF) in complex with DNA, we set up a robust computational protocol to screen the entire library and selected just a few compounds (i.e., antraquinones, ferruginines and Iboga-alkaloids, all of them containing different alkyl side chains) as possible Gli1 inhibitor lead compounds. Among them, the most active proved to be an isoflavone naturally found in the seeds of Derris glabrescens (Leguminosae), which impairs Gli1 activity by interfering with its binding to DNA.
Notably, to investigate whether the Gli1 inhibitory activity of GlaB was associated to the direct interaction of this small molecule with the transcription factor, we monitored by NMR spectroscopy the GlaB proton mono-selective relaxation rates (Rms), which prove the slowing down of the small molecule motion upon binding to a receptor. In the presence of GST-Gli1ZF-WT, a significant increase in Rms was observed for the vicinal protons H-11 and H-12 (ring B), and to a lesser extent H-15 (ring B) and H-8 (ring C). Instead, the Rms of ring A protons were not perturbed by the presence of GST-Gli1ZF-WT and very likely were not involved in binding to Gli1ZF. To emphasize the role of both Gli1 K430 and K350 residues in its transcriptional activity, we also monitored the Rms of GlaB with the GST-Gli1ZF-K340A single mutant and the GST-Gli1ZF-K340A/K350A double mutant. The binding of GlaB to Gli1ZF-K340A, Gli1ZF-K350A and Gli1ZF-K340A/K350A mutants was also investigated by molecular modeling and compared to that observed toward Gli1ZF-WT. In agreement with NMR data, K350 was highlighted as the major anchor point for GlaB within Gli1ZF. Indeed, in Gli1ZF-WT and the Gli1ZF-K340A mutant, GlaB adopted a very similar binding conformation and contacted K350 by H-bond interactions. These findings strongly support a GlaB direct inhibitory activity on Gli1 by interfering with the cooperation of both K340 and K350 for binding to DNA and transcription function, thus blunting Gli-driven gene expression.
GlaB showed a great efficacy in the inhibition of Gli-dependent MB tumor cells growth. GlaB significantly inhibited the proliferation of Ptch1+/- MB cells and also induced a striking decrease of Gli1 mRNA levels with a strength similar to GANT61. Several tumors, including MB, contain a small subset of stem-like cells with a self renewal capability (responsible for cancer cell population expansion). GlaB suppressed the ability of Ptch1+/- MB-SCs to form neurospheres from single cell suspension that appeared reduced in number and size. GlaB strikingly reduced Hh pathway activity, stemness markers (Nanog, Oct4) as well as growth (cyclins D1 andD2, PCNA) and oncogenic (NMyc) related signals. To study the GlaB effect in vivo, we turned to an allograft model of MB cells. Nude mice were grafted with spontaneous primary MB from Ptch1+/- mice and treated every second day with s.c. injections of GlaB at a concentration of 75 µmol/kg or solvent only (n = 6 for each group). During an 18-day treatment period, suppression of tumor cell growth was observed in GlaB-treated mice compared to controls. MB orthotopic xenograft animal model confirmed the inhibitory effect of GlaB to impair MB tumor growth in vivo.
This small molecule turned out to be an efficient inhibitor of the growth of Hh/Gli-dependent tumors and cancer stem cells in vitro and in vivo, indicating that Gli/DNA interference is an appealing therapeutic strategy to control the heterogeneous molecular changes leading to Hh/Gli pathway activation in cancer.
2. Total synthesis of natural compounds
· Total Synthesis of (±)-Kuwanol E
Kuwanol E is the most potent naturally occurring inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B reported so far (Ki = 1.6 ± 0.1 μM). Kuwanol E is a polyphenolic secondary metabolite isolated from the roots of Sorocea ilicifolia and from cell cultures of Morus alba and Morus nigra. In view of its biological activity, we performed the first total synthesis of (±)-kuwanol E, (±)-kuwanol E heptamethyl ether, (±)-exo-kuwanol E heptamethyl ether, and (±)-kuwanon Y heptamethyl ether. The synthetic procedure of the Diels−Alder-type adducts (±)-kuwanol E and the heptamethyl ether derivative of (±)-kuwanon Y has been accomplished via a convergent strategy involving 2′-hydroxychalcone and dehydroprenylstilbene, in nine steps. The synthesis features, as a key step, a Lewis acid-mediated biomimetic intermolecular Diels−Alder [4+2] cycloaddition for the construction of the cyclohexene skeleton with three stereogenic centers. Notably, the endo/exo diastereoselectivity of the reaction proved to be temperature-controlled.
3. Creative Chemistry
· Undecenyl resorcarene in the chair conformation as preorganized synthon for olefin metathesis
Tetramerization of (E)-2,4-dimethoxycinnamic acid ω-undecenyl ester with ethereal BF3 gave three stereoisomers, which were assigned as the chair, cone, and 1,2-alternate conformations. The chair conformation was confirmed by X-ray diffraction analysis, which also showed a peculiar self-assembly behavior in the crystal lattice, forming intercalated hydrophilic and hydrophobic layers (6–7 Å thickness) as a consequence of strong CH–π interactions. Undecenyl resorcarene, which featured the simplest pattern of substituents, was submitted to olefin metathesis using the second generation Grubbs complex as the catalyst. Depending on the reaction conditions, different products were isolated: a bicyclic alkene (46%), a linear dimer (5%), and a cross-linked homopolymer (44%).
· First detection of a ruthenium-carbene-resorcarene complex during the progress of a metathesis reaction
We describe the detection, for the first time, of a ruthenium-carbene-resorcarene complex produced, as a key intermediate, during an olefin metathesis reaction carried out on a resorcarene bicyclic olefin with [Ru(=CHPh)Cl2(PCy3)2] Grubbs first-generation (G1ST) catalyst. The complex was identified by high-resolution (600 MHz) 1H, 31P NMR and DOSY spectroscopy, in a non-invasive fashion. The singlets at 19.98 ppm (1H NMR) and 53.37 ppm (31P NMR), attributed to the carbene proton linked to ruthenium in the G1ST catalyst, were selected as probes for a kinetic analysis, and they proved to reduce at the expense of the singlets at 19.26 (1H NMR) and 52.68 ppm (31P NMR), diagnostic of the ruthenium-carbene-resorcarene complex. Such resorcarene activated olefin proved to behave as key propagating species leading to oligomers according to a ROMP pathway.
• Synthesis of a Double-Spanned-Resorcarene via Ring- Closing Metathesis and Calculation of Aggregation Propensity
Ring-closing metathesis (RCM) catalyzed by the second-generation Grubbs catalyst has been used to synthesize resorcarenes starting from undecenyl resorcarene fixed in thecone conformation. X-ray diffraction analysis revealed for the major metathesis product (50% yield) a cavity-shaped architecture resembling a basket, endowed with a large (≈ 10 A) intramolecular space, and a strong propensity to self-assembly as a supramolecular trio of heterochiral dimers. This prompted us to investigate the aggregation propensity of basket also in THF/water solution by UV-visible spectroscopy. Cavitation Gibbs free energy change (NNGcav = 4.78 kcal mol–1) associated with the self-assembly of macrocycle was calculated as a measure of the solvophobic interactions involved in the process.
4. Molecular Recognition
• Reaction of Nitrosonium Cation with Resorcarenes Activated by Supramolecular Control: Covalent Bond Formation
Resorcarenes, which previously proved to entrap NO+ cation within their cavities under conditions of host-to-guest excess, were treated with a 10-fold excess of NOBF4 salt in chloroform. Kinetic and spectral UV−visible analyses revealed the formation of isomeric 1:2 complexes as a direct evolution of the previously observed event. Accordingly, three-body 1−(NO+)2 and 2−(NO+)2 adducts were built by MM and fully optimized by DFT calculations at the B3LYP/ 6-31G(d) level of theory. Notably, covalent nitration products were obtained by reaction of NOBF4 salt with resorcarenes, respectively, involving macrocycle ring-opening and insertion of a nitro group in one of the four aromatic rings. In particular, compounds 4 and 6, both containing a trans-double bond in the place of the methine bridge, were oxidized to aldehydes 5 and 7, respectively, after addition of water to the reaction mixture. Calculation of the charge and frontier orbitals of the aromatic donor (HOMO) and the NO+ acceptor (LUMO) clearly suggests an ipso electrophilic attack by a first NO+ unit on the resorcinol ring, mediated by the second NO+ unit.
• N-Linked Peptidoresorcarene-Based Receptors as Noncompetitive Inhibitors for R-Chymotrypsin
This paper deals with the design, synthesis, and evaluation of a new series of receptors for protein surface recognition. The design of these agents is based around the attachment of four constrained dipeptide chains onto a central resorcarene scaffold. By varying the sequence, nature, and stereochemistry of the chains we prepared anionically functionalized N-linked peptidoresorcarenes by Pd/C-catalyzed hydrogenation of the corresponding benzyl esters. From this family of receptors we have identified noncompetitive inhibitors of R-chymotrypsin (ChT), which function by binding to the surface of the enzyme in the neighborhood of the active site cleft (Ki values ranging from 12.4 ± 5.1 μM for free carboxylic acid to 0.76 ± 0.14 μM for benzyl ester). For anionically functionalized receptors the ChT inhibition is based essentially on electrostatic interaction, and the bound enzyme can be released from the resorcarene surface by increasing the ionic strength, with its activity almost completely restored. For receptors with terminal benzyl ester groutg ps a hydrophobic network can be suggested.
· Structural Basis of Enzymatic (S)-Norcoclaurine Biosynthesis
The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1A˚ resolution. NCS shares no common features with the functionally correlated “Pictet-Spenglerases” that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-A˚ -long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.