Barbara Richichi

Carbonic anhydrase IX from cancer-associated fibroblasts drives epithelial-mesenchymal transition in prostate carcinoma cells

Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.

A TRPA1 antagonist reverts oxaliplatin-induced neuropathic pain

Neuropathic pain (NeP) is generally considered an intractable problem, which becomes compelling in clinical practice when caused by highly effective chemotherapeutics, such as in the treatment of cancer with oxaliplatin (OXA) and related drugs. In the present work we describe a structurally new compound, ADM_09, which proved to effectively revert OXA-induced NeP in vivo in rats without eliciting the commonly observed negative side-effects. ADM_09 does not modify normal behavior in rats, does not show any toxicity toward astrocyte cell cultures, nor any significant cardiotoxicity. Patch-clamp recordings demonstrated that ADM_09 is an effective antagonist of the nociceptive sensor channel TRPA1, which persistently blocks mouse as well as human variants of TRPA1. A dual-binding mode of action has been proposed for ADM_09, in which a synergic combination of calcium-mediated binding of the carnosine residue and disulphide-bridge-forming of the lipoic acid residue accounts for the observed persistent blocking activity toward the TRPA1 channel.

A Cancer Therapeutic Vaccine based on Clustered Tn‐Antigen Mimetics Induces Strong Antibody‐Mediated Protective Immunity

Tumor-associated carbohydrate antigens (TACAs) are key components of cancer vaccines. A variety of vaccines based on native TACAs such as α-Tn have shown immunogenicity and protection in preclinical animal studies, however, their weak immunogenicity, low in vivo instability, and poor bioavailability, have discouraged their further evaluations in clinical studies. A new improved vaccine prototype is reported. It is composed of four clustered Tn-antigen mimetics and a immunogenic peptide epitope that are conjugated to a cyclopeptide carrier. The immunization of mice with this vaccine 1) was safe, 2) induced a strong and long-lasting Tn-specific response with IgM/IgG antibodies able to recognize native carbohydrate antigens; 3) produced high titers of IgG1, IgG2a, and IgG3 antibodies; and 4) produced a significant antibody-dependent regression of tumors and conferred protection. Altogether, these findings pave the way for the clinical development of safe and effective therapeutic vaccines against Tn-expressing cancers.

Enantioselective synthesis of N, O-psiconucleosides

Abstract The first enantioselective synthesis of β- d and β- l N , O -psiconucleosides is reported. The synthetic approach is based on the asymmetric 1,3-dipolar cycloaddition of the C -[(4 S )-2,2-dimethyl-1,3-dioxolan-4-yl]- N -methyl nitrone with ethyl 2-acetyloxyacrylate followed by Vorbruggen nucleosidation, and removal of the chiral auxiliary. Stereochemical assignments are supported by a DFT theoretical study of the cycloaddition reaction.

Stereoselective synthesis of new bicyclic N,O-iso-homonucleoside analogues

The synthesis of two new bicyclic nucleoside analogues is reported. These compounds are iso-homonucleoside and are synthesised through a 1,3-dipolar cycloaddition of an enantiopure cyclic nitrone to protected allyl acohol and subsequent introduction of thymine by a Mitsunobu reaction.

A High-Affinity Carbohydrate- Containing Inhibitor of Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a class of Zn-containing hydrolases secreted by living cells and which are specialized in endopeptidase activity. MMPs participate in various biological processes such as embryonic development, wound healing, nerve growth, and angiogenesis. Aberrant MMP activities, inducing excessive degradation of the extracellular matrix, are involved in the genesis of diseases such as cancer, rheumatoid arthritis, pulmonary emphysema, and skin ulceration. Since many of these pathologies may benefit from the control of MMP activity, the quest for suitable human MMP inhibitors (MMPIs) has been actively pursued for more than a decade. Many inhibitors endowed with high affinity but modest selectivity, based on a variety of molecular scaffolds, have been reported. Some of these inhibitors have entered into clinical trials for different indications, primarily cancer and arthritis. However, high affinity is often achieved by introducing lipophilic substituents on suitable binding scaffolds, thereby decreasing solubility in water and compromising oral bioavailability. Solubility in water is required for maintaining high drug levels in plasma, which is essential for treatments relying on oral administration. Only very few of the plethora of potentially useful MMPIs reported to date are water soluble. Doses of drug higher than those based on intrinsic efficacy must be administered because of limited bioavailability. High doses, in turn, exacerbate the adverse effects of modest selectivity by causing indiscriminate inhibition of other zinc endopeptidases. Furthermore, MMP-related pathologies are usually chronic and any plausible pharmacological scheme would require longterm treatment, during which time lipophilic drugs tend to accumulate in tissues and thereby enhance side effects. High lipophilicity also increases binding affinity to human serum albumin (HSA). Strong binding to HSA has an adverse effect on bioavailability by increasing the half-life in vivo and preventing the drug from reaching the target site. In the present communication we report a new high-affinity MMP inhibitor that addresses most of the fundamental issues discussed above, using a conceptually novel strategy whereby a glycosidic residue is introduced in the appropriate location of the molecule. The inhibitor is a prototype structure that opens the way to the design of a new class of highly effective MMPIs. N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH) is one of the most prominent representatives of a family of inhibitors possessing nanomolar affinity for several MMPs (Scheme 1). The NNGH family of inhibitors suffers from the major drawbacks discussed above, and is therefore inadequate for most applications. The recently published X-ray crystallographic structure of the NNGH–MMP-12 complex allowed us to ascertain that the interaction of the inhibitor with the active site of the enzyme involves binding of the hydroxamate moiety to the catalytic Zn ion and binding of the aromatic group to the S1’ subsite (MMP-12 is the enzyme implicated in the development of emphysema). The isopropyl group on the sulfonamide nitrogen atom points away from the shallow S2’ pocket and does not directly participate in binding. In an effort to overcome the limitations of NNGH, this structural information was used to prepare the new inhibitor 1, as depicted in Scheme 1, whereby the isopropyl group was replaced with a glucosylated N-hydroxyethyl chain. The leading concept in designing 1 was to replace a portion of the molecule not directly involved in binding with a watersoluble residue, linked through a spacer of appropriate length, in the hope that its inhibiting properties would not be affected. It must be emphasized that adding a carbohydrate residue to the inhibitor is unprecedented in the NNGH family. The diasteromerically pure b anomer of compound 1 was selectively obtained in good yield (26% over five steps) by reaction of trichloroacetimidate 2 with the hydroxyethyl sulfonamide 3 under Schmidt’s glycosylation conditions. Compound 3 was obtained by treatment of the corresponding sulfonamide of the glycine methylester 4 with ethylene oxide and methyl iodide. The synthesis, which produces the glycosidic b isoACHTUNGTRENNUNGmer exclusively, has been specifically devised as a general method easily amenable to the preparation of the desired derivatives (see below) on a multigram scale through the appropriately substituted imidate. As expected, compound 1 exhibited a marked increase in water solubility (>30 mm) compared with NNGH, which is essentially insoluble. Even more significantly, the sugar moiety of structure 1 is easily changed to adjust hydrophilicity. Indeed, the solubility of 1 can be scaled down in a stepwise manner by sequentially substituting non-hydrophilic substituents [a] Dr. V. Calderone, Dr. M. Fragai, Prof. C. Luchinat, Prof. C. Nativi Magnetic Resonance Center, University of Florence via Sacconi 6, Sesto Fiorentino 50019 (Italy) Fax: (+39)0554574271 E-mail : luchinat@cerm.unifi.it [b] Prof. C. Nativi, Dr. B. Richichi Department of Organic Chemistry, University of Florence via della Lastruccia 13, Sesto Fiorentino 50019 (Italy) Fax: (+39)0554573570 E-mail : cristina.nativi@unifi.it [c] Dr. V. Calderone Department of Chemistry, University of Siena, Siena (Italy) [d] Dr. M. Fragai, Prof. C. Luchinat Department of Agricultural Biotechnology, University of Florence, Florence (Italy) [e] Prof. C. Luchinat, Dr. B. Richichi ProtEra S.r.l. , Sesto Fiorentino 50019 (Italy) [f] Dr. S. Roelens CNR—Istituto di Chimica dei Composti Organometallici, Florence (Italy) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author: synthetic procedures and characterization details ; crystallographic and NMR spectroscopic data; fluorimetric details.

Structure-based approach to nanomolar, water soluble matrix metalloproteinases inhibitors (MMPIs).

N-arylsulfonyl-based MMPs inhibitors (MMPIs) are among the most prominent inhibitors possessing nanomolar affinity. However, their poor bioavailability remains critical for the drug development of this family of molecules. The structural analysis of the complex of NNGH (the most representative member of the family) with MMP-12 provided us with the basis to effectively design simple NNGH analogues with enhanced solubility in water. Following this approach, the sec-butyl residue, not directly involved in the binding with MMP, has been replaced with hydrophilic residues thus yielding new potent inhibitors soluble in water.

Stable GM3 lactone mimetic raises antibodies specific for the antigens expressed on melanoma cells.

Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patients immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM(3) ganglioside 1 and especially the expression of its metabolite GM(3) lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM(3) lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in vivo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM(1) was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.

Design In Silico, Synthesis and Binding Evaluation of a Carbohydrate-Based Scaffold for Structurally Novel Inhibitors of Matrix Metalloproteinases

The matrix metalloproteinases (MMPs) are members of a continuously growing family of Zn-dependent endopeptidases that function extracellularly. This clan of enzymes, specialized in endopeptidase activity, is involved in both normal and pathological tissue remodeling. Activation and over-expression of MMPs seem to be connected with pathological conditions such as arthritis, cardiovascular diseases, multiple sclerosis, and cancer-cell metastasis. Since preclinical studies clearly showed that the inhibition of MMPs would be therapeutic for such diseases, the generation of effective and selective inhibitors has become an extremely attractive goal. Early approaches to the identification of potential MMP inhibitors (i.e. substrate-based design of peptidomimetics and random screening of natural product or compound libraries) have recently been replaced by structure–activity relationship (SAR) studies. Deeper insights into enzyme–ligand interactions have been possible from SAR studies, and structure elucidation through X-ray and NMR spectroscopy of MMP catalytic domain/inhibitor complexes showed that the interaction of the inhibitor with zinc in the active site is very important in determining biological potency. Beside the active-site zinc(ii) ion, all MMPs are characterized by a hydrophobic cavity, conventionally designated the S1’ pocket, which offers the greatest opportunity for selective-inhibitor design because there is considerable variation between the MMPs in its dimensions and the residues that line the pocket. Thus peptidomimetics that incorporate a zinc ligand and S1’ side chain represent one of the most common classes of MMP inhibitors (i.e. Marimastat, Batimastat), while another class of zinc ligand compounds are sulfonamide-based inhibitors such as NNGH or AG3340.

Targeting matrix metalloproteinases: design of a bifunctional inhibitor for presentation by tumour-associated galectins.

A new strategy to exploit galectin presence to target matrix metalloproteinases (MMPs) is presented. A bifunctional conjugate with lactose and an inhibitor for MMPs is able to bind MMP and Gal-3 simultaneously. This compound might allow the lectin to attract the MMP inhibitor to the tumour site and to block protumoural activities of the lectin at the same time.