Immunoglobulin free light chains in severe asthma patient: Could they be a new biomarker?

BACKGROUND: Increasing evidence is available about the presence of increased serum concentration of immunoglobulin (Ig) free light chains (FLCs) in both atopic and non-atopic inflammatory diseases, including severe asthma, providing a possible new biomarker of disease. METHODS: We analyzed clinical and laboratory data, including FLCs, obtained from a cohort of 79 asthmatic subjects, clinically classified into different GINA steps. A control group of 40 age-matched healthy donors (HD) was considered. Particularly, HD have been selected according to the absence of monoclonal components (in order to exclude paraproteinemias), were tested for total IgE (that were in the normal ranges) and were negative for aeroallergens specific IgE. Moreover, no abnormality of common inflammatory markers (i.e., erythrocyte sedimentation rate and C-reactive protein) was detectable. RESULTS: FLC-k levels were significantly increased in the asthmatic population, compared to the control group. Despite the absence of statistically significant differences in FLC-λ levels, the FLC-k/FLC-λ ratio displayed remarkable differences between the two groups. A positive correlation between FLC-κ and FLC-λ levels was found. FLC- λ level displayed a significant negative correlation with the FEV1 value. Moreover, the FLC-κ /FLC- λ ratio was negatively correlated with the SNOT-22 score and a positive correlation was observed between FLCs and Staphylococcus Aureus IgE enterotoxins sensitization. CONCLUSIONS: Our findings confirmed the role of FLCs in asthma as a potential biomarker in an inflammatory disease characterized by different endotypes and phenotypes. In particular, FLC-κ and FLC-k/FLC-λ ratio could be a qualitative indicator for asthma, while FLC-λ levels could be a quantitative indicator for clinical severity parameters.

Plants and Small Molecules: An Up-and-Coming Synergy.

The emergence of Arabidopsis thaliana as a model system has led to a rapid and wide improvement in molecular genetics techniques for studying gene function and regulation. However, there are still several drawbacks that cannot be easily solved with molecular genetic approaches, such as the study of unfriendly species, which are of increasing agronomic interest but are not easily transformed, thus are not prone to many molecular techniques. Chemical genetics represents a methodology able to fill this gap. Chemical genetics lies between chemistry and biology and relies on small molecules to phenocopy genetic mutations addressing specific targets. Advances in recent decades have greatly improved both target specificity and activity, expanding the application of this approach to any biological process. As for classical genetics, chemical genetics also proceeds with a forward or reverse approach depending on the nature of the study. In this review, we addressed this topic in the study of plant photomorphogenesis, stress responses and epigenetic processes. We have dealt with some cases of repurposing compounds whose activity has been previously proven in human cells and, conversely, studies where plants have been a tool for the characterization of small molecules. In addition, we delved into the chemical synthesis and improvement of some of the compounds described.

Toxicological aspects of cannabinoid, pesticide and metal levels detected in light Cannabis inflorescences grown in Italy.

Recently, the cultivation of light Cannabis, with a total THC content less than 0.6%, has been encouraged due to its industrial and therapeutic potential. This has increased the consumption of hemp for both smoking purposes and food preparation. Even so, Cannabis inflorescences are not subject to EU regulations and standards provided for food and tobacco products. A study was carried out on thirty-one inflorescences samples, collected in different Italian regions, in order to determine cannabinoids, pesticides and metals and to evaluate the exposure of consumers to contaminants and ensure a safe consumption. Contents of THC were always below 0.5%, while CBD ranged between 0.3 and 8.64%. The determination of 154 pesticides showed that 87% of the samples contained fungicides and insecticides in the range 0.01-185 µg/g. The most found are spinosad and cyprodinil. The concentration of metals ranged from 1 to more than 100 µg/g and As, Cd, Co, Cr, Hg, Cu, Mo, Ni and V exceeded the regulatory US limits for inhaled Cannabis products, while Pb exceeded them for both oral and inhaled products. These contaminants are intrinsically toxic and may affect public health. Actions are needed to establish regulatory measures and reduce the adverse effects caused by contaminants in Cannabis.

Curcumin-loaded graphene oxide flakes as an effective antibacterial system against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml-1. Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.

Diketo acids derivatives as dual inhibitors of human immunodeficiency virus type 1 integrase and the reverse transcriptase RNase H domain.

The HIV-1 integrase (IN) and reverse transcriptase (RT) are essential enzymes in the virus cycle. RT is crucial for the retrotranscription of the RNA viral genome, while IN is involved in the insertion in host chromosome of the proviral double strand DNA produced by RT. This enzyme has two associated functions: the RNA- and DNA-dependent DNA polymerase (RDDP and DDDP) and the ribonuclease H (RNase H). The RNase H function catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate. Since the discovery that catalytic cores of both HIV-1 RNase H and IN are folded in a very similar way, have very similar active site geometries, and show the same DDE triad absolutely required for catalytic activity, some researches were devoted to study IN and RNase H dual inhibitor. Our decennial interest in design and synthesis of IN inhibitors led us to study the activity of our compounds also on RNase H activity. The results of the activities showed by pyrrolyl and quinolonyl diketo acids are reported and discussed.

HIV-1 RT-associated RNase H function inhibitors: Recent advances in drug development.

The HIV-1 genomic RNA reverse transcription is an essential step in the virus cycle carried out by the viral-coded reverse transcriptase (RT), which has two associated functions: the RNA- and DNA-dependent DNA polymerase (RDDP and DDDP) function and the ribonuclease H (RNase H) function. The RNase H function catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate. The RT associated activities are both essential for HIV-1 replication and validated targets for drug development, but only the polymerase function has been widely investigated as drug target. In fact, either nucleoside or non-nucleoside RT inhibitors currently used in therapy act on the polymerase associated activity. In this review, we describe the compounds, reported up to today, which inhibit the HIV-1 RNase H function, their chemical structures, the structure-activity relationships and the mechanism of action.

Competing sigmatropic shift rearrangements in excited allyl radicals.

The competition between rearrangement of the excited allyl radical via a 1,3 sigmatropic shift versus sequential 1,2 shifts has been observed and characterized using isotopic substitution, laser excitation, and molecular beam techniques. Both rearrangements produce a 1-propenyl radical that subsequently dissociates to methyl plus acetylene. The 1,3 shift and 1,2 shift mechanisms are equally probable for CH(2)CHCH(2), whereas the 1,3 shift is favored by a factor of 1.6 in CH(2)CDCH(2). The translational energy distributions for the methyl and acetylene products of these two mechanisms are substantially different. Both of these allyl dissociation channels are minor pathways compared to hydrogen atom loss.

Design, synthesis and biological evaluation of heteroaryl diketohexenoic and diketobutanoic acids as HIV-1 integrase inhibitors endowed with antiretroviral activity.

Highly active anti-retroviral therapy (HAART) using reverse transcriptase (RT) and protease (PR) inhibitors and, more recently, inhibitors of the fusion is currently the best clinical approach in combating acquired immunodeficiency syndrome (AIDS), caused by infection from human immunodeficiency virus type 1 (HIV-1). However, this therapy does not completely eradicate the virus, so that resistant strains easily emerge. The above problem calls urgently for research on inhibitors of further viral targets such as integrase (IN), the third enzyme produced by HIV. Recently, our research group was engaged in studies on conformationally restrained cinnamoyl compounds related to curcumin as anti-IN agents. Compounds containing both a 3,4,5-trihydroxyphenyl group and a carboxylic acid function were potent IN inhibitors active against viral replication. More recently, a promising new class of inhibitors synthesized by Merck Company has emerged, which contain aryldiketoacid (ADK) functionality. The ADKs selectively inhibited the stand transfer (ST) step of integration and were proven to be effective IN inhibitors in vivo. Our interest in the field of IN inhibitors led us to design pyrrole and indole derivatives containing both a cinnamoyl moiety and a diketoacid group. A number of the cited derivatives were proven potent IN inhibitors, which selectively inhibited the ST step at submicromolar concentrations and were effective against virus replication in HIV-1 infected cells.

Enantioselective liquid chromatography of C3-chiral 2,3-dihydro-1,2,5-benzothiadiazepin-4(5H)-one and thione 1,1-dioxides on polyacrylamide- and polysaccharide-based chiral stationary phases.

Optically active synthetic and semisynthetic polymers were utilized as chiral stationary phases (CSPs) for the direct chromatographic enantioseparation of a series of 8-chloro-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5H)-one and thione 1,1-dioxide. Evaluation of stereochemical integrity of chiral analytes was assessed by enantioselective temperature and flow-dependent HPLC. A stopped-flow high-performance liquid chromatography (sfHPLC) procedure was developed for the determination of the rate constants and free energy barriers of enantiomerization of enantiomers of 8-chloro-2-(3-methylbut-2-enyl)-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5H)-thione 1,1-dioxide (compound 2) in the presence of Chiraspher and Chiralcel OD CSPs. In order to study the chiroptical properties of the individual enantiomers of analytes investigated, semipreparative chromatographic resolutions were performed. The assignment of the absolute configuration was empirically established by comparing the CD spectra of the separated enantiomers with those obtained from structural analogues.

Conversion of a racemic mixture of 8-chloro-2-(2,6-difluorophenylmethyl)-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5h)-one 1,1-dioxide into a single enantiomer via a chromatographic resolution/racemization method.

A simple and efficient strategy to convert the racemic mixture of 8-chloro-2-(2,6-difluorophenylmethyl)-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5H)-one 1,1-dioxide, a new anti-HIV-1 agent targeted to reverse transcriptase, into the more active (S)-enantiomer is described. The method utilizes repetition of the following two steps: 1) semipreparative enantioseparation by HPLC on chiral stationary phase; 2) base-induced racemization of the less active (R)-enantiomer.

Analytical and semipreparative enantiomeric separation of azole antifungal agents by high-performance liquid chromatography on polysaccharide-based chiral stationary phases. Application to in vitro biological studies.

High-performance liquid chromatography (HPLC) was used for the enantiomeric separation of chiral imidazole derivatives endowed with antimycotic activity. Enantioselective columns, containing carbamates of cellulose and amylose, were used. The influence of the nature and content of an alcoholic modifier in the mobile phase was studied. The isolated enantiomers, separated on semipreparative columns, were submitted to in vitro biological investigations.

Antimycobacterial pyrroles: synthesis, anti-Mycobacterium tuberculosis activity and QSAR studies.

A number of known antifungal pyrrole derivatives and some newly synthesized compounds (5-33) were tested in vitro against Mycobacterium tuberculosis CIP 103471. The majority of tested compounds were efficient antimycobacterial agents showing MIC values ranging from 0.5 to 32 microg/mL. A 3-D-QSAR study has been performed on these pyrrole derivatives to correlate their chemical structures with their observed inhibiting activity against M. tuberculosis. Due to the absence of information on a putative receptor responsible for this activity, classical quantitative structure-activity relationships (QSAR) and comparative molecular field analysis (CoMFA) have been applied. A model able to well correlate the antimycobacterial activity with the chemical structures of pyrrole derivatives 5-33 has been developed which is potentially helpful in the design of novel and more potent antituberculosis agents. The combination of CoMFA with classical QSAR descriptors led to a better hybrid 3-D-QSAR model, that successfully explains the structure-activity relationships (r2 = 0.86) of the training set. A comparison between the QSAR, CoMFA and mixed QSAR-CoMFA models is also presented. The hybrid model is to be preferred, however, because of its lowest values of the average absolute error of prediction toward a limited external test set.

Structure-activity relationship studies on potential non-nucleoside DABO-like inhibitors of HIV-1 reverse transcriptase.

Using 2,6-dichloro-4-aminopyrimidine, a number of uracil and cytosine derivatives with both arylthio and alkoxy moieties were prepared. These novel pyrimidines share chemical similarities with DABOs and HEPTs, two classes of non-nucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitors (NNRTIs), which have been widely studied of late. All new derivatives were tested in MT-4 cells to explore their potential in vivo anti-HIV activity. Like other NNRTIs, they selectively inhibit HIV-1 but not HIV-2. The majority of test derivatives were found to have low potency and were sometimes more cytotoxic than zidovudine and emivirine (formerly MKC-442), used here as reference drugs. Uracil and cytosine derivatives bearing a sec-butoxy chain and a methyl-substituted benzenesulphonyl moiety were the most potent. Enzyme assays proved that these derivatives target RT. Structure-activity relationship studies established a correlation between the anti-HIV-1 activity and the meta substitution on the phenyl ring; furthermore, oxidation of sulphide to sulphone significantly increased the potency of certain derivatives.

Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling.

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2, 4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1, 3-indandione, and others. While active against both 3'-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the cinnamoyl residue C=C-C=O in a syn disposition, differently from flavone derivatives characterized by an anti arrangement about the same fragment. Certain inhibitors, lacking one of the two pharmacophoric catechol hydroxyls, retain moderate potency thanks to nonpharmacophoric fragments (i.e., a m-methoxy group in curcumin) which favorably interact with an "accessory" region of IN. This region is supposed to be located adjacent to the binding site accommodating the pharmacophoric dihydroxycinnamoyl moiety. Disruption of coplanarity in the inhibitor structure abolishes activity owing to poor shape complementarity with the target or an exceedingly high strain energy of the coplanar conformation.

1,2,5-Benzothiadiazepine and pyrrolo[2,1-d]-[1,2,5]benzothiadiazepine derivatives with specific anti-human immunodeficiency virus type 1 activity.

We synthesized and tested as novel inhibitors of human immunodeficiency virus type 1 (HIV-1) bi- and tricyclic thiadiazine ring homologues of 7-chloro-2-ethyl-2H-1,2,4-benzothiadiazin-3-(4H)-one 1,1-dioxide, which is a compound endowed with anti-HIV-1 activity at low micromolar concentrations. Benzothiadiazepine derivatives were obtained by alkylation of 8-chloro-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5H)-one 1,1-dioxide, which was obtained by intramolecular cyclization of 2-(2-amino-5-chloro-benzenesulphonamido) propanoic acid. Pyrrolobenzothiadiazepines were synthesized from N-substituted 5-chloro-2-(1H-pyrrol-1-yl)benzene-sulphonamides by treating with triphosgene. N6-substituted pyrrolo[2,1-d][1,2,5]benzothiazepin-7(6H)-one 5,5-dioxides were active, though not very potent. Both a chlorine atom and an unsaturated alkyl chain were found to be determinants of anti-HIV-1 activity. The highest potency was shown by a derivative with a TIBO-related 3,3-dimethylallyl chain. 2,3-Dihydro-1,2,5-benzothiadiazepin-4(5H)-one 1,1-dioxides were scarcely active in HIV-1-infected MT-4 cell assays; however, the introduction of the dimethylallyl chain into 7-chloro-1,2,5-benzothiadiazepine moiety led to a bicyclic derivative which was more potent and less cytotoxic than the tricyclic pyrrole-containing counterpart.

Pyrrolnitrin and related pyrroles endowed with antibacterial activities against Mycobacterium tuberculosis.

During development of nitroheterocycles with potential antimycobacterial activities we have tested against Mycobacterium tuberculosis a number of pyrroles strictly related to pyrrolnitrin, an antifungal antibiotic isolated from Streptomyces pyrrocinia. Some of the tested arylpyrrole derivatives and pyrrolnitrin have shown appreciable inhibiting activity against M. tuberculosis and M. avium. SAR studies well correlate antimycobacterial potency with the presence of halogens in the phenyl ring and a nitro group at position 3 of pyrrole.

Heterocycles with a benzothiadiazepine moiety. 5. Derivatives of pyrrolo[2,1-d][1,2,5]benzothiadiazepine, a novel tricyclic ring.

The synthesis of pyrrolo[2,1-d][1,2,5]benzothiadiazepin-7(6H)-one 5,5-dioxide has been achieved by reaction between 2-(1H-pyrrol-1-yl)benzenesulfonamide and triphosgene. N-Ethylation of the tricyclic derivative afforded 6-ethylpyrrolo[2,1-d][1,2,5] benzothiadiazepin-7(6H)-one 5,5-dioxide, also obtained by the action of trifosgene on N-ethyl 2-(1H-pyrrol-1-yl)benzenesulfonamide. Preparation of pyrrole derivatives from 2-aminobenzenesulfonamide and its N-ethyl derivative by Clauson-Kaas procedure required preliminary protection of the sulfonamide function.

Molecular modeling of azole antifungal agents active against Candida albicans. 1. A comparative molecular field analysis study.

A series of 56 azole antifungal agents belonging to chemically diverse families related to bifonazole, one of the antimycotic drugs of clinical use, were investigated using the comparative molecular field analysis (CoMFA) paradigm. The studied compounds, which have been already synthesized and reported to be active in vitro against Candida albicans, were divided into a training set and a test set. The training set consisted of 40 molecules from all the different structural classes. Due to the lack of experimental structural data on these derivatives, molecular mechanics techniques were used to obtain putative active conformations for all the compounds. the correctness of this molecular modeling work was confirmed a posteriori by comparison with structural data of the analog 2w obtained by X-ray crystallographic analysis (Massa, S.; et al. Eur. J. Med. Chem. 1992, 27, 495-502). Two different alignment rules of the training set molecules were used in this study and are based on the assumption that according to published results on azole antifungal agents, all the studied compounds exert their inhibitory activity through the coordination of their azole moiety to the protoporphyrin iron atom of the fungal lanosterol 14alpha-demethylase enzyme. The predictive ability of each resultant CoMFA model was evaluated using a test set consisting of 16 representative compounds that belong to all the different structural classes. The best 3D-quantitative structure-activity relationship model found yields significant cross-validated, conventional, and predictive r2 values equal to 0.57, 0.95, and 0.69, respectively. The average absolute error of predictions of this model is 0.30 log units, and the structural moieties of the studied antifungal agents which are thought to contribute to the biological activity were identified. The predictive capability of this model could be exploited in further synthetic studies on antifungal azoles. Furthermore, the results obtained by using two different alignments of the inhibitors suggest that the binding mode of these molecules involves both a coordination to the iron protoporphyrin atom and an additional, likewise relevant, hydrophobic interaction with the active site.

Antifungal agents. 9. 3-Aryl-4-[alpha-(1H-imidazol-1-yl)arylmethyl]pyrroles: a new class of potent anti-Candida agents.

A new class of potent antifungal agents, namely, 3-aryl-4-[alpha-(1H-imidazol-1-yl)arylmethyl]-pyrroles, is described. These compounds are related to bifonazole and pyrrolnitrin, two compounds belonging to the class of antimycotic drugs. The synthesis of the title pyrroles has been performed starting from 1,3-diaryl-2-propen-1-ones, which were reacted with tosylmethyl isocyanide to give 3-aroyl-4-arylpyrroles. Reduction of the resulting compounds by lithium aluminum hydride furnished the related alcohols, which were treated with 1,1'-carbonyldimidazole to afford the required imidazole derivatives. Forty-four new pyrroles which incorporate an (arylmethyl)imidazole moiety in the 3-arylpyrrole structure were prepared by the above procedure and tested in vitro against Candida albicans and Candida spp. Among test compounds, 10 were found to be highly active against C. albicans. The most active derivative (27) was twice as potent (MIC90) as bifonazole, and its activity was 4 times greater than those of miconazole and ketoconazole. The other nine compounds showed antifungal activity of the same order of that of bifonazole and were ca. 2 times as active as miconazole and ketoconazole. Derivatives 21 and 27 tested in vivo against C. albicans A170 were shown to be highly effective in rabbit skin candidosis. Pharmacological studies on compounds 27 and other related pyrroles (19, 35, 36, 38, 39, and 49) are in progress to select one of them as a potential candidate for clinical experiments.

Antifungal agents. VIII. Synthesis and antifungal activities of bipyrryl analogues of bifonazole.

Various bipyrryl analogues of bifonazole were synthesized starting from aryl-3-pyrryl-1-imidazolylmethanes. The introduction of a second pyrryl portion was performed by linking an acrylate moiety at 1-position of the pyrrole ring and then by treatment with TosMIC. The bipyrryl esters were hydrolyzed and decarboxylated to afford the required imidazoles. All new imidazole derivatives were tested against Candida albicans and Candida spp using as standard controls miconazole, bifonazole and ketoconazole.