Fluorescence method for determining the mechanism and speed of action of surface-active drugs on yeast cells

New antifungal agents are needed to treat life-threatening fungal infections, particularly with the development of resistance. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a time-dependent drug exposure assay that allows us to rapidly investigate the mechanism of surface-active antifungal drug action. The assay uses a multidrug pump-deficient strain of Saccharomyces cerevisiae and the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide [diS-C?(3)] and can assess whether cells are depolarized, hyperpolarized, or permeabilized by drug exposure. In this work, we investigated the mechanisms of action of five surface-active compounds: SDS, nystatin, amphotericin B, octenidine dihydrochloride, and benzalkonium chloride. The diS-C?(3) time-dependent drug exposure assay can be used to identify the mechanisms of action of a wide range of drugs. It is a fast and cost-effective method for screening drugs to determine their lowest effective concentrations.     

The mechanism of action of inhibitors of bacterial two-component signal transduction systems

Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs.     

Targeting chemotherapy-resistant tumour sub-populations using inorganic chemistry: Anti-cancer stem cell metal complexes

Cancer stem cells (CSC) are a sub-population of tumours linked to metastasis and relapse. Current chemotherapeutic drug options are ineffective against CSCs at their administered doses. New families of cytotoxic agents, and new, highly specific ways of delivering them to CSCs, are needed to provide durable clinical outcomes. Inorganic compounds have recently emerged as a promising class of anti-CSC agents with clinically relevant potencies. In this short review, we present the very latest efforts (post-2020) on the development of anti-CSC metal complexes. The activities of the metal complexes in monolayer and three-dimensional CSC cultures and animal models is documented. The mechanism of action of the metal complexes with respect to their chemical structures is also highlighted.     

Effects of subacute treatment with benzopyranopyrimidines in hemostasis and experimental thrombosis in mice

The antithrombotic activity of a series of benzopyranopyrimidine derivatives was investigated in platelet-dependent and independent pulmonary thromboembolism in mice. Intraperitoneal subacute treatment with 2-morpholino derivative 3c significantly prevented paralysis due to collagen plus epinephrine-induced pulmonary thrombosis while 2-piperidino substituted derivative 3h significantly protected mice from paralysis caused by thrombin-induced intravascular fibrin formation at dosage not affecting bleeding time. These compounds, previously proved to be effective as antiplatelet agents in vitro, were in vivo more potent as antithrombotics than lysine acetylsalicylate and possessed lower prohemorrhagic activity than the reference drug. Although their ineffectiveness on clotting times, PT and APTT, allows the involvement of coagulation pathways to be ruled out, the mechanisms underlying the favourable benefit risk ratio for these two compounds remain to be further clarified.     

Synthesis, antiplatelet and antithrombotic activities of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones

Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipyridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-aggregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones and the results of the pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.     

Antiarrhythmic drug therapy: new drugs and changing concepts

The effective treatment of life-threatening ventricular arrhythmias (VA) leading to sudden cardiac death remains a major health problem. Cellular electrophysiological techniques that have provided insight into the underlying mechanisms of these arrhythmias have also provided a convenient classification of antiarrhythmic drugs based on their dominant electrophysiological action. Traditional pharmacological approaches to the management of VA have involved primarily class I agents. Newer drugs in this class are potent conduction depressants (class IC agents), which, however, have been limited in their clinical impact on VA because of unwanted cardiac and extracardiac side effects. Other recent approaches include the introduction of class III agents, which are thought to interrupt primarily reentrant impulses by specific prolongation of action potential duration and refractoriness without compromising normal cardiac conduction. Newer approaches may also include drugs with greater specificity of action, agents with combinations of electrophysiological effects (class I/III, I/II), drugs exemplifying novel mechanisms of action such as anion antagonism (class V), and agents controlling sympathetic neural outflow. The growing awareness of the potential proarrhythmic effects of antiarrhythmic drugs has also become important in drug development and assessment, as emphasized by the search for improved methods of drug selection (e.g., programmed electrical stimulation). Finally, the desired characteristics of a new antiarrhythmic agent are presented as a goal for future drug development.     

New anthelmintics

The anthelmintic drugs currently available are still relatively few in number. Many of them are limited in their usefulness because of narrow spectrum of action, safety, high cost or impractical delivery systems. Some of these will be discontinued because of unforseen problems such as occurrence of drug resistance which needs more attention than it presently receives.Drug development is costly and time consuming and requires exacting interdisciplinary interactions between researchers using the most stringent quantitative tests to determine toxicity, safety, mode of action and pharmacokinetics before a drug is ever used in clinical trials. A system of universal testing standards accepted by all involved in drug-development should be established. Scientists are beginning to apply rational design to the development of new compounds and this approach can be expected to be exploited more in the future.From the clinical standpoint, the future is beginning to look brighter for the millions of persons suffering from helminth diseases as a result of available broad spectrum compounds which can be used in clinics and mass-treatment programs.     

Replicative Homeostasis III: implications for antiviral therapy and mechanisms of response and non-response

While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies.     

Pharmacologically active metabolites,combination screening and target identification-driven drug repositioning in antituberculosis drug discovery

There has been renewed interest in alternative strategies to address bottlenecks in antibiotic development. These include the repurposing of approved drugs for use as novel anti-infective agents, or their exploitation as leads in drug repositioning. Such approaches are especially attractive for tuberculosis (TB), a disease which remains a leading cause of morbidity and mortality globally and, increasingly, is associated with the emergence of drug-resistance. In this review article, we introduce a refinement of traditional drug repositioning and repurposing strategies involving the development of drugs that are based on the active metabolite(s) of parental compounds with demonstrated efficacy. In addition, we describe an approach to repositioning the natural product antibiotic, fusidic acid, for use against Mycobacterium tuberculosis. Finally, we consider the potential to exploit the chemical matter arising from these activities in combination screens and permeation assays which are designed to confirm mechanism of action (MoA), elucidate potential synergies in polypharmacy, and to develop rules for drug permeability in an organism that poses a special challenge to new drug development.     

Zebrafish model of photochemical thrombosis for translational research and thrombolytic screening in vivo

Acute thromboembolic diseases remain the major global cause of death or disability. Although an array of thrombolytic and antithrombotic drugs has been approved to treat or prevent thromboembolic diseases, many more drugs that target specific clotting mechanisms are under development. Here a novel zebrafish model of photochemical thrombosis is reported and its prospective application for the screening and preclinical testing of thrombolytic agents in vivo is demonstrated. Through photochemical excitation, a thrombus was induced to form at a selected section of the dorsal aorta of larval zebrafish, which had been injected with photosensitizers. Such photochemical thrombosis can be consistently controlled to occlude partially or completely the targeted blood vessel. Detailed mechanistic tests indicate that the zebrafish model of photochemical thrombosis exhibits essential features of classical coagulation and a thrombolytic pathway. For demonstration, tissue plasminogen activator (tPA), a clinically feasible thrombolytic agent, was shown to effectively dissolve photochemically induced blood clots. In light of the numerous unique advantages of zebrafish as a model organism, our approach is expected to benefit not only the development of novel thrombolytic and antithrombotic strategies but also the fundamental or translational research targeting hereditary thrombotic or coagulation disorders.

     

Identification of cryptotanshinone from Tongmai to inhibit thrombosis in zebrafish via regulating oxidative stress and coagulation cascade

BackgroundThromboembolic events are leading causes of mortality and morbidity all over the world. Tongmai (TM) is a botanical drug with valid clinical efficacy and safety in the management of thrombosis and ischemic cardiovascular diseases, however, its active compounds and underlying mechanism are largely unclear.PurposeTo investigate the endogenous effects, therapeutic mechanism and active compounds of TM in thrombus formation.Study designCombined with transgenic zebrafish models and high-content imaging system, this study evaluated the endogenous antithrombotic effects of TM and screened for the active compounds.MethodsThe PHZ-induced thrombotic model in erythrocytes or platelets labeled transgenic zebrafish were established, to dynamically evaluate the antithrombotic effects of TM. The oxidative damage levels were analyzed by specific fluorescent probes, and the expression levels of key factors in coagulation cascades and platelet activation were examined by QPCR. TM were dissected into fractions by reverse phase chromatography and subsequently screened for their antithrombotic effects in the transgenic fish models. The compounds of the active TM fraction were then analyzed by UPLC-Q-TOF analysis and further verified for their antithrombotic effects and mechanisms.ResultsIn PHZ-induced zebrafish thrombotic model, TM incubation markedly increased cardiac blood flow, decreased peripheral erythrocytes aggregation, and recovered peripheral platelet circulation. Besides, the levels of oxidative stress and lipid peroxidation were increased in the PHZ-induced thrombotic fish, which were greatly decreased by TM treatment. Moreover, TM significantly reduced the expression of coagulation factor II (thrombin) and the downstream fibrinogen. In order to identify the active compounds of TM, four fractions were separated from the extract by reverse phase chromatography, which were subsequently screened for their antithrombotic effects in the fish model. As a result, fraction 4 showed the strongest effect in inhibiting thrombosis. Finally, through UPLC-Q-TOF analysis and endogenous screening, cryptotanshione was identified as the main active compound with antithrombotic effects.ConclusionOur study demonstrated the endogenous antithrombotic effects of TM, which is possibly mediated by inhibiting oxidative stress and coagulation cascade. Cryptotanshione was identified as a major compound with antithrombotic activity and is a promising candidate for novel antithrombotic therapy.     

THE EFFECT OF NEW DRUGS ON THE RHYTHMIC FUNCTION OF THE HEART

Epinephrine and related drugs (sympathomimetic amines) are the only compounds which effectively increase the rhythmic function of the heart.Effects of two new non-pressor sympathomimetic compounds were observed. One of these compounds, the isopropyl homologue of epinephrine, was found to be about five times more active than epinephrine.From clinical observations on the action of a new cardiac depressant drug, alpha-fagarine, it was concluded that the drug is very effective but that toxic reactions are unpredictable.The potency of alpha-fagarine and related compounds suggests the possible development of chemically related non-toxic drugs having an effective cardiac depressant action.     

Synthesis of novel 3-carboxamide-benzocoumarin derivatives as orally active antithrombotic agents

In an effort to develop potent antithrombotic agents, a series of novel 3-carboxamide-benzocoumarin derivatives were synthesized and screened for their in vivo antithrombotic activity. Among 20 compounds tested, the compound 12b showed the most promising antithrombotic activity which was comparable with clinically used aspirin or warfarin, but, at variance with these standard drugs, 12b did not exhibit the increased bleeding time, suggesting its potential as a novel antithrombotic agent.     

Thrombin inhibitors with lipid peroxidation and lipoxygenase inhibitory activities

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagulant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in the treatment of thrombosis. Of the compounds 10a-h and 11a-d, shown to possess thrombin inhibitory activity, 11a-d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity makes compounds 10 and 11 interesting candidates for further investigations towards multiple antithrombotic drugs.     

Discovery and development of SQ109: a new antitubercular drug with a novel mechanism of action

Existing drugs have limited efficacy against the rising threat of drug-resistant TB, have significant side effects, and must be given in combinations of four to six drugs for at least 6 months for drug-sensitive TB and up to 24 months for drug-resistant TB. The long treatment duration has led to increased patient noncompliance with therapy. This, in turn, drives the development of additional drug resistance in a spiral that has resulted in some forms of TB being currently untreatable by existing drugs. New antitubercular drugs in development, particularly those with mechanisms of action that are different from existing first- and second-line TB drugs, are anticipated to be effective against both drug-sensitive and drug-resistant TB. SQ109 is a new TB drug candidate with a novel mechanism of action that was safe and well tolerated in Phase I and early Phase II clinical trials. We describe herein the identification, development and characterization of SQ109 as a promising new antitubercular drug.     

Inhibition of platelet aggregation and thrombosis by indole alkaloids isolated from the edible insect Protaetia brevitarsis seulensis (Kolbe)

Protaetia brevitarsis seulensis (Kolbe) has been temporarily registered as a food material by the Ministry of Food and Drug Safety of Korea (MFDS). The current study aimed to discover small antithrombotic molecules from this edible insect. Five indole alkaloids, 5‐hydroxyindolin‐2‐one ( 1 ), (1R,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 2 ), (1S,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 3 ), (3S)‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 4 ) and L‐tryptophan ( 5 ), were isolated from the insect. Among them, compounds 1 and 2 prolonged aPTT and PT and impaired thrombin and FXa generation on HUVEC surface. Moreover, these compounds inhibited platelet aggregation. Antithrombotic effects of compounds 1 and 2 were further confirmed in pre‐clinical models of pulmonary embolism and arterial thrombosis. Collectively, these results demonstrated that compounds 1 and 2 could be effective antithrombotic agents and serve as new scaffolds for the development of antithrombotic drug.     

A 2-sulfated, 3-linked alpha-L-galactan is an anticoagulant polysaccharide

Marine alga is an abundant source of sulfated polysaccharides with potent anticoagulant activity. However, several attempts to identify the specific structural features in these compounds, which confer the biological activity, failed due to their complex, heterogeneous structure. We isolated and characterized several sulfated alpha-L-galactans and sulfated alpha-L-fucans from marine invertebrates. In contrast to the algal fucans and galactans, these invertebrate polysaccharides have a simple structure, composed of well-defined units of oligosaccharides. We employed two of these compounds to elucidate their structure-anticoagulant action relationship. Our results indicate that a 2-sulfated, 3-linked alpha-L-galactan, but not an alpha-L-fucan, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaces thrombin. Thus, the anticoagulant activity of sulfated galactan and sulfated fucan is not merely a consequence of their charge density. The interaction of these polysaccharides with coagulation cofactors and their target proteases are specific. Identification of specific structural requirements in sulfated galactans and sulfated fucans necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.     

Reinforcement schedules and extrapolations to humans from animals in behavioral pharmacology.

Behavior controlled by various schedules of reinforcement is useful for characterizing drugs as well as for analyzing the mechanisms of action of their effects on behavior. Conditioned avoidance techniques have been useful for studying neuroleptics and for predicting their clinical antipsychotic acitivity; the possible involvement of dopaminergic mechanisms in the effect of neurolpetics on avoidance behavior is discussed. Tricyclic antidepressant agents have been studied in assays involving interactions with other agents, such as cocaine, amphetamine and tetrabenazine. One type of operant behavior, Sidman avoidance, has been used as particularly sensitive assay for such drug interactions. Another schedule, in which "observing" responses in pigeons are measured. seems to provide a method for studying antidepressants without involving drug interaction phenomena. For tricyclic compounds, facilitation of observing responses and weak potency of conditioned avoidance inhibition constitute a pharmacological profile that seems to have some predictive value for clinical imipramine-like antidepressant activity. "Conflict (punishment) schedules have been useful for predicting antianxiety activity in man. Although the degree of anticonflict effect observed is consistent with Dew's rate dependency hypothesis, this principle does not fully account for the observed drug effects. In the conflict model, the actions of benzodiazepines differ in drug-naive versus drug-experienced animals. Experiments with parachlorophenylalnine have not yet provided clear support for the postulated role of serotonin in related phenomena.     

Medicinal mushrooms and cancer therapy: translating a traditional practice into Western medicine

Modern medical practice relies heavily on the use of highly purified pharmaceutical compounds whose purity can be easily assessed and whose pharmaceutical activity and toxicity show clear structure-function relationships. In contrast, many herbal medicines contain mixtures of natural compounds that have not undergone detailed chemical analyses and whose mechanism of action is not known. Traditional folk medicine and ethno-pharmacology coupled to bioprospecting have been an important source of many anticancer agents as well as other medicines. With the current decline in the number of new molecular entities from the pharmaceutical industry, novel anticancer agents are being sought from traditional medicine. As the example of medicinal mushrooms demonstrates, however, translating traditional Eastern practices into acceptable evidence-based Western therapies is difficult. Different manufacturing standards, criteria of purity, and under-powered clinical trials make assessment of efficacy and toxicity by Western standards of clinical evidence difficult. Purified bioactive compounds derived from medicinal mushrooms are a potentially important new source of anticancer agents; their assimilation into Western drug discovery programs and clinical trials also provides a framework for the study and use of other traditional medicines.     

Deciphering the molecular mechanisms of anti-tubulin plant derived drugs

Even though commercialized anticancer drugs are now produced by pharmaceutical companies, most of them were originally obtained from natural sources, and more particularly from plants. Indeed, many structurally diverse compounds isolated from plants or marine flora have been purified and synthesized for their anticancer bioactivity. Among these, several molecules belong to the class of anticancer drugs which target the microtubule cytoskeleton, either by stabilizing it or destabilizing it. To characterize the activity of these drugs and to understand in which physiological context they are more likely to be used as therapeutic agents, it is necessary to fully determine their interaction with tubulin. Understanding the molecular basis of their effects on microtubule cytoskeleton is an important step in designing analogs with greater pharmacological activity and with fewer side effects. In addition, knowing the molecular mechanism of action of each drug that is already used in chemotherapy protocols will also help to find strategies to circumvent resistance. By taking examples of known anti-tubulin plant derived drugs, we show how identification of microtubule targeting agents and further characterization of their activity can be achieved combining biophysical and biochemical approaches. We also illustrate how continuing in depth study of molecules with already known primary mechanisms of action can lead to the discovery of new targets or biomarkers which can open new perspectives in anticancer strategies.