Metal coordination and peripheral substitution modulate the activity of cyclic tetrapyrroles on αS aggregation: a structural and cell-based study.

The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein.

A new iridoid, verbascoside and derivatives with inhibitory activity against Taq DNA polymerase.

DNA polymerases are enzymes that play a crucial role in DNA metabolism such as replication, repair, transcription, recombination, and chromosome segregation during mitosis. Herein we report the isolation of a new iridoid (6-epi-catalpol, 2) and per-O-acetyl-verbascoside (11) from aerial part of Buddleja cordobensis Grisebach (Buddlejaceae). From compound 2, we have obtained eight compounds by chemical transformation. This group of compounds at a concentration of 500µM was assayed against Taq DNA polymerase. Compound 11 (per-O-acetyl-verbascoside) was the most active with an IC50 of 1.21±0.18µM; compounds 9, 2 and 8 were strong inhibitors with IC50 values of 5.57±0.70, 21.62±0.22 and 78.13±0.93µM, respectively. Compounds 11 and 9 could be a leader structures to development new anticancer chemotherapy medicines and a useful tool to investigate DNA polymerase activity.

Inhibition of reverse transcriptase and Taq DNA polymerase by compounds possessing the coumarin framework.

Coumarin derivatives were prepared using natural products isolated from plants belonging in the Pterocaulon genus (Asteraceae) and commercial drugs. Some molecules have displayed interesting activity against myeloid murine leukemia virus-reverse transcriptase (MMLV-RT) (compounds 20 and 28 produced inhibition with IC50 values of 38.62 and 50.98 µM, respectively) and Taq DNA polymerase (analogues 13 and 14 produced inhibition with IC50 values of 48.08 and 57.88 µM, respectively). Such inhibitors may have importance as antiretroviral chemotherapeutic agents and also in the development of anticancer drugs.

A reappraisal on lidocaine-sensitive repetitive, uniform atrial tachycardia.

BACKGROUND: Lidocaine sensitive, repetitive atrial tachycardia is an unusual arrhythmia whose electrophysiologic substrate remains undefined. We aimed to analyze the electropharmacologic characteristics of this arrhythmia with emphasis on its cellular substrate and response to drug challenges. METHODS: We retrospectively analyzed a series of 18 patients from an electrocardiographic and electrophysiologic perspective and the response to pharmacological challenge. RESULTS: There was no evidence of structural heart disease in 12 patients, 4 patients presented with systemic hypertension; one patient had a prior myocardial infarction and one a mitral valve prolapse. The arrhythmia depicted a consistent pattern in nine patients. The first initiating ectopic beat showed a long coupling interval, the cycle length of the second atrial ectopic beat presented the shortest cycle length and a further prolongation was apparent towards the end of the atrial salvos. Conversely, in the other nine cases, the atrial tachycardia cycle length was erratic. The arrhythmia was suppressed by asynchronous atrial pacing at cycle lengths longer than those of the atrial tachycardia. Intravenous lidocaine eliminated the arrhythmia in all patients, but intravenous verapamil suppressed the atrial tachycardia in only two patients while adenosine caused a transient disappearance in 2/8 patients. Only one patient responded to all the three agents. Radiofrequency ablation was successfully performed in 10 patients. CONCLUSIONS: Repetitive uniform atrial tachycardia can be sensitive to lidocaine. In few cases, this rare focal arrhythmia may be also suppressed by adenosine and/or verapamil, which suggests a diversity of electrophysiologic substrates that deserve to be accurately identified.

Diuretic activity of Euphorbia serpens extracts in Wistar rats.

Euphorbia serpens has been used in central-west region of Argentina in traditional medicine as diuretic plant. The aim of this present study was to evaluate the diuretic activity of E. serpens in-vivo. We used dried aerial parts, and infusions from these were orally administered to Wistar rats. Its effect was evaluated using furosemide as a positive drug and isotonic salt solution as negative control. Their urine output was quantified at several time intervals. The volume of urine excreted and Na+ increased significantly, being similar to furosemide. Mannitol, was the main component in aqueous extracts of E. serpens, and the acetone extract showed the presence of Δ12- oleanane-type triterpenoids compounds, mainly hederagenin. No toxic effects were observed.

Synthesis of Structurally Related Coumarin Derivatives as Antiproliferative Agents.

A library of structurally related coumarins was generated through synthesis reactions and chemical modification reactions to obtain derivatives with antiproliferative activity both in vivo and in vitro. Out of a total of 35 structurally related coumarin derivatives, seven of them showed inhibitory activity in in vitro tests against Taq DNA polymerase with IC50 values lower than 250 µM. The derivatives 4-(chloromethyl)-5,7-dihydroxy-2H-chromen-2-one (2d) and 4-((acetylthio)methyl)-2-oxo-2H-chromen-7-yl acetate (3c) showed the most promising anti-polymerase activity with IC50 values of 20.7 ± 2.10 and 48.25 ± 1.20 µM, respectively. Assays with tumor cell lines (HEK 293 and HCT-116) were carried out, and the derivative 4-(chloromethyl)-7,8-dihydroxy-2H-chromen-2-one (2c) was the most promising, with an IC50 value of 8.47 µM and a selectivity index of 1.87. In addition, the derivatives were evaluated against Saccharomyces cerevisiae strains that report about common modes of actions, including DNA damage, that are expected for agents that cause replicative stress. The coumarin derivatives 7-(2-(oxiran-2-yl)ethoxy)-2H-chromen-2-one (5b) and 7-(3-(oxiran-2-yl)propoxy)-2H-chromen-2-one (5c) caused DNA damage in S. cerevisiae. The O-alkenylepoxy group stands out as that with the most important functionality within this family of 35 derivatives, presenting a very good profile as an antiproliferative scaffold. Finally, the in vitro antiretroviral capacity was tested through RT-PCR assays. Derivative 5c showed inhibitory activity below 150 µM with an IC50 value of 134.22 ± 2.37 µM, highlighting the O-butylepoxy group as the functionalization responsible for the activity.

Aromaticity at position 39 in α-synuclein: A modulator of amyloid fibril assembly and membrane-bound conformations.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Verbascoside, synthetic derivatives and other glycosides from Argentinian native plant species as potential antitumoral agents.

A phytochemical study was performed on three native plant species from the central-western zone of Argentina: Buddleja cordobensis Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta cataria L. We could obtain verbascoside (1) from B. cordobensis. From N. cataria, we could obtain 1, 5, 9-epi-deoxyloganic acid (2) L. Finally, we could isolate 2-ß-(L-rhamnopyranosyl)-3-angeloyloxy-15-acetyloxy-7,13(14)-E-dien-ent-labdane (3) and 2-ß-(L-rhamnopyranosyl)-3-α-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane (4) from B. salicina. Moreover, three derivatives from 1, and one semi-synthetic derivative from 2, were prepared. PCR reaction was used to analyse the activity against DNA polymerase and cell culture to determine cytotoxicity and antitumoral activity. Verbascoside (1) was strongly active in the nanomolar scale (IC50 = 356 nM) against DNA polymerization. Moreover, verbascoside was also strongly active in the nanomolar scale against human melanoma cell line (IC50 = 256 nM) and human colorectal cell line (IC50 = 320 nM). Furthermore, derivatives 6 and 7 were cytotoxic against both cancer cell lines.

DNA Related Enzymes as Molecular Targets for Antiviral and Antitumoral Chemotherapy. A Natural Overview of the Current Perspectives.

BACKGROUND: The discovery of new chemotherapeutic agents still remains a continuous goal to achieve. DNA polymerases and topoisomerases act in nucleic acids metabolism modulating different processes like replication, mitosis, damage repair, DNA topology and transcription. It has been widely documented that Polymerases serve as molecular targets for antiviral and antitumoral chemotherapy. Furthermore, telomerase is a ribonucleoprotein with exacerbated activity in most of the tumor cell lines, becoming as an emergent target in Cancer treatment. METHODS: We undertook an exhaustive search of bibliographic databases for peer-reviewed research literature related to the last decade. The characteristics of screened bibliography describe structure activity relationships and show the principal moieties involved. This work tries to summarize the investigation about natural and semi-synthetic products with natural origin with the faculty to inhibit key enzymes that play a crucial role in DNA metabolism. RESULTS: Eighty-five data references were included in this review, showing natural products widely distributed throughout the plant kingdom and their bioactive properties such as tumor growing inhibitory effects, and anti-AIDS activity. CONCLUSION: The findings of this review confirm the importance to find new drugs and biologically active natural products, and their potential medicinally useful benefits.

Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features.

The identity of the Cu(i) binding ligands at Met-X3-Met site of AcαS and its role into the affinity and structural properties of the interaction were elucidated by NMR spectroscopy. We provide evidence that the source of ligands for Cu(i) binding to the Met-X3-Met site comes from the N-terminal acetyl group and the Met-1, Asp-2 and Met-5 residues. From the study of site-directed mutants and synthetic peptide models of αS we demonstrated the critical role played by Met-1 and Met-5 residues on the binding affinity of the Cu(i) complex, acting as the main metal anchoring residues. While having a more modest impact in the affinity features of Cu(i) binding, as compared to the Met residues, the N-terminal acetyl group and Asp-2 are important in promoting local helical conformations, contributing to the stabilization of these structures by favoring Cu(i) binding.

Supernormal conduction in the anomalous bundles of the Wolff-Parkinson-White syndrome: an overlooked electrophysiologic property with potential clinical implications.

The anterograde refractory period (RP) of the accessory pathway (AP) is the main determinant factor of ventricular rate during atrial fibrillation in the Wolff-Parkinson-White (WPW) syndrome. We describe 3 examples of anterograde supernormal conduction (SNC) and 1 of retrograde SNC in APs. The paradoxical early recovery of propagation due to SNC, well inside a prolonged anterograde RP in the AP, may play a relevant role to determine the rate of ventricular response during atrial fibrillation, eventually leading to extremely fast ventricular rates, syncope, and even ventricular fibrillation in patients with WPW syndrome supposed a priori to be exposed to a low risk of sudden cardiac death. This may require very precise conditions, including an enhanced adrenergic influence on the heart. Retrograde SNC in APs may also participate in the mechanism of paroxysmal supraventricular tachycardias that are not easily induced by programmed cardiac stimulation.

Masquerading bundle branch block obscuring the diagnosis of Brugada syndrome: an electrocardiographic and vectorcardiographic study.

We describe the induction of a masquerading bundle branch block in two patients with Brugada syndrome following the administration of Ajmaline. The development of this conduction disturbance prevented the correct electrocardiographic diagnosis. However, the simultaneously obtained vectocardiogram identified both the Brugada pattern and the masquerading bundle branch block.

Inappropriate sinus tachycardia may be related to an immunologic disorder involving cardiac beta andrenergic receptors.

BACKGROUND: The mechanisms underlying inappropriate sinus tachycardia are not fully known. An autonomic imbalance seems to play a role, but no attempts have been made to investigate a relationship between this arrhythmia and the antiautonomic membrane receptor antibodies found in other heart disorders and arrhythmias. OBJECTIVE: The purpose of this study was to investigate the prevalence and the functional and biochemical effects of circulating antiautonomic receptor antibodies in patients with inappropriate sinus tachycardia. METHODS: We studied 21 patients with inappropriate sinus tachycardia and 15 healthy volunteers. The chronotropic effects of the IgG fractions (also of affinity-purified anti-beta1 adrenergic receptor antibodies in selected cases) were assessed on cultured cardiomyocytes before and after exposure to atropine and propranolol. The effects of the IgG fractions from five patients and five healthy volunteers on cAMP production were evaluated in COS-7 cells transfected with genes encoding for beta1 or beta2 adrenergic receptor. RESULTS: The IgG fractions from patients with inappropriate sinus tachycardia exerted a positive chronotropic action with a high prevalence of anti-beta receptor antibodies (52%) and induced a clear-cut and long lasting increment of cAMP. No anti-M2 cholinergic receptor antibodies were found. The IgG fractions from healthy volunteers did not contain antiautonomic receptor antibodies. CONCLUSIONS: Our results suggest, for the first time, a link between inappropriate sinus tachycardia and circulating anti-beta adrenergic receptor antibodies that induce a persistent increment in cAMP production. This finding offers new insight into the physiopathology of inappropriate sinus tachycardia with potential therapeutic consequences.

Recording of high V1-V3 precordial leads may be essential to the diagnosis of Brugada syndrome during the ajmaline test.

Sodium channel-blocking agents are routinely used to unveil the Brugada syndrome in patients in whom the typical electrocardiographic pattern is absent or doubtful. In this article, the authors report a patient with syncopal episodes of unknown origin in whom the conventional electrocardiographic result was normal and a negligibly small "saddle back" type repolarization was present in lead V2 recorded 2 intercostal spaces above the conventional site. Intravenous ajmaline (50 mg) did not elicit the type 1 pattern of the Brugada syndrome in the precordial leads obtained at their usual level, but a clear-cut coved-type repolarization was apparent in high right precordial leads. These findings indicate that high precordial leads should be routinely recorded while assessing the ajmaline test in patients suspected of having the Brugada syndrome.

Cytotoxic and Antitumor Activity of some Coumarin Derivatives.

Several natural and synthetic coumarins were assayed against different cancer cell lines. Four of them have shown cytotoxicity against a panel of three human solid tumor cell lines (HeLa, T-47D, and WiDr) and a clearly activity/hydrophobicity relationship. Compound 13 proved to be the most active product in all cell lines tested, with values of 8.0 (±0.38) µM against HeLa cells and also able to inhibit Taq DNA polymerase. This dual activity of 13 makes it a candidate to be considered as a "lead" compound in the search for novel antitumor drugs.

Coumarins as Potential Inhibitors of DNA Polymerases and Reverse Transcriptases. Searching New Antiretroviral and Antitumoral Drugs.

Human Immunodeficiency Virus (HIV) is the viral agent of Acquired Immunodeficiency Syndrome (AIDS), and at present, there is no effective vaccine against HIV. Reverse Transcriptase (RT) is an essential enzyme for retroviral replication, such as HIV as well as for other RNA infectious viruses like Human T lymphocyte virus. Polymerases act in DNA metabolism, modulating different processes like mitosis, damage repair, transcription and replication. It has been widely documented that DNA Polymerases and Reverse Transcriptases serve as molecular targets for antiviral and antitumoral chemotherapy. Coumarins are oxygen heterocycles that are widely distributed throughout the plant kingdom. Natural coumarins have attraction due to their bioactive properties such as tumor promotion inhibitory effects, and anti-HIV activity. Coumarins and derivates exhibit potent inhibitory effects on HIV-1 replication in lymphocytes and compounds isolated from Calophyllum inophyllum or DCK derivates showed inhibitory activity against human RT. Furthermore, natural isocoumarins isolated from cultures of fungi or hydroxycoumarins were able to inhibit human DNA polymerase. In view of their importance as drugs and biologically active natural products, and their medicinally useful properties, extensive studies have been carried out on the synthesis of coumarin compounds in recent years. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), a class of antiretroviral chemotherapeutic agents, act by binding to an allosteric pocket showing, generally, low toxicity. This work tries to summarize the investigation about natural and synthetic coumarins with the ability to inhibit key enzymes that play a crucial role in DNA metabolism and their possible application as antiretroviral and antitumoral agents.

The mechanisms of spontaneous termination of reentrant supraventricular tachycardias.

BACKGROUND: Atrioventricular nodal reentrant tachycardia (AVNRT) and atrioventricular reentrant tachycardia (AVRT) often terminate spontaneously, presumably due to changes in the electrophysiological properties of the reentrant circuit. However, the mechanism of spontaneous termination of these arrhythmias is incompletely understood. METHODS: We included 70 consecutive patients with reentrant supraventricular tachycardias (35 AVNRT, 35 AVRT) in whom the arrhythmia ended spontaneously during the electrophysiologic study. We determined in each patient the duration of the induced arrhythmia, site of block, beat-to-beat oscillations in tachycardia cycle-length (CL), A-H, H-V, H-A and V-A intervals. RESULTS: In 21/34 (62%) patients with AVNRT and 19/30 (63%) with orthodromic AVRT, tachycardia termination was preceded by progressive increase in tachycardia CL due to prolongation of the A-H interval (Mobitz type-I pattern). In 13/34 patients with AVNRT (38%) and 11/30 with orthodromic AVRT (37%), termination occurred suddenly without a preceding change in CL, with block ensuing retrogradely either in the fast AV nodal pathway or the accessory pathway (Mobitz type-II pattern). In 4/5 patients with antidromic AVRT the tachycardia ended at the retrograde limb with previous prolongation of the VA interval. CONCLUSION: Spontaneous termination of AVNRT and AVRT is a time-related phenomenon. Despite different pathways being involved in these two reentrant tachycardias, termination can follow antegrade or retrograde block in similar ratio (60% antegradely and 40% retrogradely). Antegrade block is preceded by prolongation of the AH interval (Mobitz type-I), whereas retrograde block occurs unexpectedly in the retrograde limb (Mobitz type-II). Fatigue of conduction appears to be involved in this phenomenon.

Chemistry and biological activity of coumarins at molecular level.

Synthetic coumarins were prepared in high yields using ionic liquids as an environmental friendly alternative. 3,4-Dimethyl-7-hydroxycoumarin (3ab) and 3-isopropyl-4-methyl-5,7-dihydroxycoumarin (3bc) showed interesting activity against Taq DNA polymerase with IC50 values of 115.7 microM and 82.2 microM, respectively. Also, 4-methyl-7-hydroxycoumarin (3aa) and 4-methyl-5,7-dihydroxycoumarin (3ba) exhibited inhibitory activity against MMLV-RT with IC50 values of 23.5 microM and 18.3 microM, respectively. These inhibitors could have importance as antiretroviral chemotherapeutic agents and also for the development of antitumor drugs.