Paola Milla

Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in α-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 50% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

Inhibition of bovine β-trypsin, human α-thrombin and porcine pancreatic β-kallikrein-B by 4′,6-diamidino-2-phenylindole, 6-amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study

Abstract The inhibitory effect of 4′,6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine β-trypsin (trypsin), human α-thrombin (thrombin) and porcine pancreatic β-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 ± 0.5°C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin: DAPI complex was solved at 1.9 A resolution ( R = 0.161). Over the whole pH range explored, values of the association inhibition constant ( K i ) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic p K shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin: DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

Rationally designed inhibitors as tools for comparing the mechanism of squalene-hopene cyclase with oxidosqualene cyclase

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OCS) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

Intravesical thermo-chemotherapy based on conductive heat: a first pharmacokinetic study with mitomycin C in superficial transitional cell carcinoma patients.

PurposeTo evaluate, for the first time, the mitomycin C (MMC) pharmacokinetics during intravesical hyperthermia treatment based on conductive heat and the stability and recovery of the drug at the end of the instillation period.MethodsEleven patients with recurrent intermediate-risk superficial transitional cell carcinoma of the bladder were treated weekly for six cycles with intravesical MMC (40xa0mg MMC in 50xa0ml) in local hyperthermia (45xa0°C) with Unithermia® system. Each instillation lasted 45xa0min, with the solution being replaced after the first 22xa0min. The MMC recovery at the end of the two instillation period and the plasmatic pharmacokinetics of MMC were evaluated by high-pressure liquid chromatography.ResultsNine patients completed all the six planned cycles, whereas two patients missed the last cycle because of allergic reactions. No other systemic toxicity was observed, and the local toxicities were mild. Median MMC concentration in the instillation residual solution decreases from the initial 0.8 to 0.22xa0mg/ml for the 0–22-min instillation period and to 0.38xa0mg/ml for the 22–45-min instillation period; the median percentage of MMC recovered after instillation was 66.2 and 99.6, respectively. In all patients, MMC plasmatic Cmax resulted considerably lower than the toxic threshold (400xa0ng/ml).ConclusionsThe MMC is stable during the instillation, and its absorption occurs mainly during the first minutes of the treatment. The plasmatic MMC concentration is always well below the threshold level for myelosuppression, as confirmed by the total lack of hematological toxicity evidenced by the patients. In order to evaluate the efficacy of the treatment performed with UniThermia® in reducing the disease recurrence rate in short- and long-term follow-up, we are currently carrying out a clinical multicentric study involving a larger number of patients.

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae.

Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas’ disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Using a homogenate of S. cerevisiae cells expressing T. cruzi OSC, we have tested 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, selected as representative of different classes of substrate analog inhibitors of OSC. The IC50 values of inhibition (the compound concentration at which the enzyme is inhibited by 50%) are compared with the values obtained using OSC of pig liver and S. cerevisiae. Many inhibitors of pig liver and S. cerevisiae OSC show comparable IC50 for T. cruzi OSC, but some phenylthiovinyl derivatives are 10–100 times more effective on the T. cruzi enzyme than on the pig or S. cerevisiae enzymes. The expression of proteins of pathogenic organisms in yeast seems very promising for preliminary screening of compounds that have potential therapeutic activity.

Stereospecific syntheses of trans-vinyldioxidosqualene and β-hydroxysulfide derivatives, as potent and time-dependent 2,3-oxidosqualene cyclase inhibitors

trans-Vinyldioxidosqualene and beta-hydroxysulfide derivatives were synthesized stereospecifically and evaluated as inhibitors of animal and yeast oxidosqualene cyclases. Only trans-vinyldioxidosqualene and 2,3-epoxy-vinyl-beta-hydroxysulfides, having the reactive function at crucial positions 14,15 and 18,19, were active as inhibitors of animal and yeast cyclases. (14-trans)-28-Methylidene-2,3: 14,15-dioxidoundecanorsqualene 27 was the most potent inhibitor of the series of pig liver cyclase, with an IC50 of 0.4 microM, and it behaved also as the most active time-dependent inhibitor of the animal enzyme.

Preparation and characterization of novel poly(ethylene glycol) paclitaxel derivatives.

Paclitaxel has been found to be very effective against several human cancers; one of the major problems with its use is its poor solubility, which makes necessary its solubilization with excipients that can determine allergic reactions often severe. The aim of this study is to develop highly water-soluble prodrugs of paclitaxel. For this purpose we prepared a series of new paclitaxel-poly(ethylene glycol) (PEG) conjugates that were characterized and evaluated for their in vitro stability and cytotoxicity. In particular, in order to modulate the release of paclitaxel from prodrugs, we prepared different compounds introducing PEG in the drug C2 and/or C7 positions via ester or carbamate linkage. The conjugates were obtained in high purity and good yield. The carbamate prodrugs were highly stable in different media, while the compounds obtained linking PEG at C2 position through an ester bond showed lower stability. Finally, the cytotoxic activity of the conjugates was evaluated on two cancer cell lines and the results showed that all the derivatives had a reduced cytotoxicity compared to that of paclitaxel.

Active site titration of bovine β-trypsin by Nα-(N,N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis

Kinetics of bovine β‐trypsin (trypsin) with the N α ‐(N,N‐dimethylcarbamoyl)‐α‐aza‐lysine p‐nitrophenyl ester (Dmc‐azaLys‐ONp) was obtained at pH 6.2 and 21.0°C. Dmc‐azaLys‐ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three‐dimensional structure of the trypsin · N α ‐(N‐N‐dimethylcarbamoyl)‐α‐aza‐lysine acyl · enzyme adduct has been solved by X‐ray crystallography at 2.0 Å resolution (R = 0.145). The Dmc‐azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue.

Subcutaneous vs. intravenous immunoglobulin in CIDP: pharmacokinetic and clinical response

Dear Editor, Subcutaneous immunoglobulin (SCIg) represents an innovative and effective alternative to intravenous immunoglobulin (IVIg) for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (Cocito et al., 2014; 2015). Different tolerability profiles have been reported between SCIg and IVIg, with the SCIg possible advantages of fewer side effects and greater independence from hospital care (Markvardsen et al., 2013; Rajabally, 2014). However, the two routes of administration are associated with a distinctive pharmacological profile (Eftimov et al., 2009), which may result in a differential clinical efficacy. We compare the pharmacokinetic data of eight CIDP patients shifted from IVIg monthly infusions to SCIg weekly administrations, evaluating the possible correlations between clinical response and IgG plasma levels. All patients were receiving IVIg for at least 6 months, reporting a “wear-off effect” between each infusion (increase of≥ 1 point at the inflammatory neuropathy cause and treatment [INCAT] score and/or decrease of≥1 point at the medical research council [MRC] scale). IVIg monthly dose (average dose: 1.25± 0.38 g/ kg/month; ranging from 1 to 2 g/kg/month, according to the individual clinical response) was converted to an equivalent dose of SCIg (20% solution of immunoglobulin ready-to-use) delivered via a programmable pump at the patient’s domicile. The shift from IVIg to SCIg was due to fluctuations in clinical response (wear-off) and patient’s desire of greater independence from hospital care (IVIg administration at the patient’s domicile is not allowed in Italy). Blood samples (5 ml), clinical scales (INCAT and MRC) and Martin vigorimeter measurements were collected immediately before the first day (T-1) and after the second day (T0) of the last IVIg cycle, and at the following time-points: (1) before the first SCIg administration (2 weeks after the last IVIg

Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody

The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half‐life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD‐1) or reciprocally swapped (DCD‐2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met‐mediated biological responses were inhibited, including anchorage‐dependent and ‐independent cell growth. In vivo DCD‐1 and DCD‐2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half‐life and reducing the clearance. In pre‐clinical models of cancer, generated by injection of tumor cells or implant of patient‐derived samples, systemic administration of the engineered molecules inhibited the growth of Met‐addicted tumors.