HIV-1 Integrase Inhibition Activity by Spiroketals Derived from Plagius flosculosus, an Endemic Plant of Sardinia (Italy) and Corsica (France)

In this work we investigated, for the first time, the effect of Plagius flosculosus (L.) Alavi & Heywood, a Sardinian–Corsican endemic plant, on HIV-1 integrase (IN) activity. The phytochemical analysis of the leaves chloroform extract led us to isolate and characterize three compounds (SPK1, SPK2, and SPK3) belonging to the spiroketals, a group of naturally occurring metabolites of phytochemical relevance with interesting biological properties. Due to their structural diversity, these cyclic ketals have attracted the interest of chemists and biologists. SPK1, SPK2, and SPK3 were evaluated here for their ability to inhibit HIV-1 integrase activity in biochemical assays. The results showed that all the compounds inhibited HIV-1 IN activity. In particular, the most active one was SPK3, which interfered in a low molecular range (IC50 of 1.46 ± 0.16 µM) with HIV-1 IN activity in the presence/absence of the LEDGF cellular cofactor. To investigate the mechanism of action, the three spiroketals were also tested on HIV-1 RT-associated Ribonuclease H (RNase H) activity, proving to be active in inhibiting this function. Although SPK3 was unable to inhibit viral replication in cell culture, it promoted the IN multimerization. We hypothesize that SPK3 inhibited HIV-1 IN through an allosteric mechanism of action

Chelation motifs affecting metal-dependent viral enzymes: N′-acylhydrazone ligands as dual target inhibitors of HIV-1 Integrase and Reverse Transcriptase Ribonuclease H domain

Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N′-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N′-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes

Chemical Characterization and Anti-HIV-1 Activity Assessment of Iridoids and Flavonols from Scrophularia trifoliata

Plants are the everlasting source of a wide spectrum of specialized metabolites, characterized by wide variability in term of chemical structures and different biological properties such antiviral activity. In the search for novel antiviral agents against Human Immunodeficiency Virus type 1 (HIV-1) from plants, the phytochemical investigation of Scrophularia trifoliata L. led us to isolate and characterize four flavonols glycosides along with nine iridoid glycosides, two of them, 5 and 13, described for the first time. In the present study, we investigated, for the first time, the contents of a methanol extract of S. trifoliata leaves, in order to explore the potential antiviral activity against HIV-1. The antiviral activity was evaluated in biochemical assays for the inhibition of HIV-1Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) activity and HIV-1 Integrase (IN). Three isolated flavonoids, rutin, kaempferol-7-O-rhamnosyl-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside, 8–10, inhibited specifically the HIV-1 IN activity at submicromolar concentration, with the latter being the most potent, showing an IC50 value of 24 n

Chelation motifs affecting metal-dependent viral enzymes: N'-acylhydrazone ligands as dual target inhibitors of HIV-1 integrase and reverse transcriptase ribonuclease H domain

Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N'-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N'-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes.status: publishe

Effetto del trattamento della celiachia in pazienti diabetici-celiaci di età pediatrica

Il diabete e la celiachia sono due patologie autoimmuni che, sovente, possono associarsi nello stesso soggetto. Il 6% dei pazienti con diabete mellito tipo 1, infatti, può sviluppare la celiachia. Scopo del nostro lavoro è stato quello di studiare l’effetto della celiachia e del suo trattamento sui parametri auxologici e sull’equilibrio glico-metabolico in pazienti con diabete e celiachia paragonati a pazienti con solo diabete. Il BMI e la media annuale dell’emoglobina glicosilata (HbA1c) e della dose insulinica sono stati rilevati in 50 pazienti (25 F e 25 M) (Gruppo A) in buon compenso metabolico (HbA1c<8,5%) di età compresa tra 17,1+6,1 e con diabete insorto all’età di 6,2+4 anni dal momento della diagnosi di celiachia (T0) posta sulla base della positività dei markers autoimmuni e della biopsia duodenale e ogni anno per 4 anni (T1,T2,T3 e T4). Tali parametri sono stati, quindi, paragonati a quelli di 50 pazienti, con solo diabete (Gruppo B), in buon compenso metabolico (HbA1c < 8,5%) paragonabili per età, sesso, durata del diabete (età 16,8+6,7 durata 10+3 aa). Nel Gruppo A si è assistito ad un aumento lineare dello z-score del BMI (incremento medio z-score 0,2) dal T0 al T4 (t student per dati appaiati <0,005) cosa che non è avvenuta nel Gruppo B. Il Gruppo A rispetto al Gruppo B ha mostrato una dose insulinica significativamente superiore a ciascun tempo (p<0,005). Sia nel Gruppo A che nel Gruppo B non si è rilevato un aumento dell’HbA1c nel tempo ed i valori dei due gruppi non erano significativamente diversi. L’aumento del BMI e della dose insulinica nei pazienti affetti da entrambe le patologie potrebbero essere dovuti, almeno all’inizio del trattamento con dieta senza glutine, al migliorato assorbimento intestinale secondario alla correzione del danno mucosale. Tuttavia tale incremento si mantiene nel tempo e la differenza tra celiaci e non celiaci è ancora significativa dopo 4 anni. Ciò potrebbe dipendere dal fatto che i prodotti senza glutine forniscono un apporto calorico superiore a quelli glutinati. Inoltre contengono più carboidrati per metabolizzare i quali è necessaria una dose insulinica maggiore. L’aumentato assorbimento, l’aumentato intake di carboidrati, l’aumentata insulinizzazione ed, infine, l’aumentato intake energetico possono spiegare i risultati ottenuti.E’ auspicabile che i progressi della ricerca merceologica possano portare alla formulazione di preparati senza glutine che siano più adatti a pazienti con diabete

Cosa possono nascondere le chetoacidosi ?

B.P.P, maschio,12 anni. Giunge alla nostra osservazione con storia di febbre, dolore addominale, poliuria, polidipsia, calo ponderale e riscontro di leucocitosi neutrofila (WBC 29.500/mmc), e iperglicemia (480 mg/dl). E.O.: paziente molto sofferente con sensorio obnubilato,respiro di Kussmaul e dolore addominale spontaneo e provocato dalla palpazione, soprattutto in sede epigastrica,glicemia 795 mg/dl,chetonemia 5,9 mmol/l.Si inizia terapia della chetoacidosi. Gli esami di laboratorio evidenziano inoltre iperamilasemia (amilasi tot 687 U/l;amilasi pancreatica 575 U/l) e iperlipasemia (1066 U/l).Si aggiunge perciò in terapia gabesato metilato e si programma di ritardare la successiva rialimentazione. Dopo 24 ore la chetonemia è normalizzata ma il paziente è ancora parzialmente obnubilato,presenta due episodi di vomito e persiste dolore in sede epigastrica sia spontaneo che alla palpazione. Nei giorni successivi vi è progressivo miglioramento clinico e laboratoristico dell’interessamento pancreatico,con piena normalizzazione in quarta giornata dal ricovero.L’alimentazione è stata reintrodotta progressivamente a partire dalla terza giornata dal ricovero. Parallelamente,la creatinina plasmatica,normale al ricovero(0,87 mg/dl),presentava un progressivo aumento (in seconda giornata 1,62 mg/dl; picco in terza giornata 3,13 mg/dl) e normalizzazione solo (0.78 mg/dl) in decima giornata.L’aumento della creatinina si accompagnava ad aumento della pressione arteriosa (valore massimo riscontrato:PAS 144 PAD 94 mmHg),il che rendeva necessaria terapia con amplodipina. La pressione arteriosa,come la creatininemia,si normalizzava in decima giornata. L’associazione di chetoacidosi diabetica,pancreatite acuta ed insufficienza renale transitoria non era mai stata osservata prima nella nostra pur ampia casistica. Nel nostro caso si esclude che il diabete sia secondario alla pancreatite (positività dei markers autoimmuni, HbA1c > 14 % già alla diagnosi.). E’ probabile che l’intensa iperlipolisi in corso di chetoacidosi diabetica induca danno della parete delle cellule pancreatiche con successiva attivazione intraparenchimale degli enzimi digestivi e conseguente “ autodigestione”. L’ IRA ,nel nostro caso,potrebbe essere di natura pre-renale per grave disidratazione o renale per NTA per shock ischemico,ipotesi diagnostiche in accordo con il normalizzarsi del pattern renale parallelamente al ristabilimento delle condizioni di idratazione del paziente

Multi target mode of HIV-1 inhibition by the bioactive molecule Lupeol extracted from the Indian plant Hemidesmus indicus R.Br.

In the effort of identifying novel therapeutic approaches for the discovery of new anti-infective drugs, the traditional Indian medicine and the use of natural compounds active against various diseases, including viral infections, receive nowadays considerable attention. Hemidesmus indicus R.Br. is known for its ethno-botanical use and its efficacy in the treatment of bone-loss diseases, and, in the Indian traditional medicine, is widely used for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever and diarrhea. In the last thirty years, many natural substances have shown antiviral activity, but very few of them showed multi target mode of action. For this reason, we assessed the Hemidesmus indicus R.Br. decoction against i) the Human Immunodeficiency Virus type 1 (HIV -1) Reverse Transcriptase (RT)-associated functions and ii) the \u3b1-glucosidase activity, with the aim to identify a molecule capable to inhibit with a multiple mode of action different promising targets. Hemidesmus indicus R.Br. decoction has been shown to be active on the inhibition of both RT-associated functions and \u3b1-glucosidase activity. Active decoction was fractionated up to obtain the bioactive components which have been isolated, tested and identified as Lupeol, Lupeol Acetate, Caffeic Acid, Chlorogenic Acid and \u3b2-amirine acetate. Among these, Lupeol inhibited the HIV-1 RT -associated RNase H function, and the \u3b1 -glucosidase activity. Our results suggest that this bioactive molecule is a potential lead for further development of drugs with multi target mechanism of action

Not Autoimmune Diabetes Mellitus in Paediatrics

The rooted conviction according to which the childhood diabetes should only be an autoimmune diabetes is gradually disappearing, thanks to the discovery of not autoimmune paediatric diabetes. Many of these forms of diabetes are a direct consequence of the mutation of a single gene, involved into the insulin secretion mechanism of the pancreatic beta cells. Mutations in genes directly involved in insulin secretion or that limit the ability of the pancreas to produce insulin are the cause of the major non-autoimmune diabetes forms of the paediatric age (monogenic diabetes). The monogenic diabetes accounts for 1–2 % of all cases of diabetes in the paediatric-adolescent age. The apparent increasing in its prevalence is probably due to the improvement of the diagnostic methods. Nowadays there have been described 13 forms of MODY, the mitochondrial diabetes, the secondary diabetes and the syndromic diabetes. In this chapter, all these forms and the differential diagnosis of them have been described with the exception of secondary diabetes and diabetes due to syndromes

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 lM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre