Despite being present in many drugs, guanylhydrazones and semicarbazones are two functional groups that have been little investigated as potential therapeutic strategies for the treatment of Alzheimer's disease (AD). For this reason, we initiated the synthesis and evaluation of these compounds as potential anticholinesterase agents, aiming to offer new alternatives for drug development against AD. In the severe phase of AD butyrylcholinesterase (BChE) becomes the main enzyme responsible for the hydrolysis of acetylcholine (ACh). Therefore, in this project, we present the results of BChE inhibitory activity, enzyme kinetics, cytotoxicity, and molecular modeling studies for three guanylhydrazone and two semicarbazone derivatives that were previously synthesized and evaluated as acetylcholinesterase (AChE) inhibitors. Among the compounds tested, guanylhydrazones (1, 2, and 3) showed inhibitory activity against BChE, exhibiting a mixed non-competitive inhibition profile. Specifically, compound 2 (phenanthrenequinone) demonstrated superior inhibitory potency with an IC50 of 0.68 μM, compared to compound 1 (acridinone) with an IC50 of 3.87 μM, and compound 3 (benzodioxole) with an IC50 of 101.7 μM. In contrast, semicarbazones (4 and 5) showed no BChE inhibition up to the highest concentration tested (300 μM). Importantly, all five compounds were found to be non-cytotoxic. Our results suggest that these compounds have potential as drug prototypes targeting different phases of AD. Compounds 3, 4, and 5 may be more effective in the early phase, when AChE activity remains high; compound 1 could be useful in the intermediate phase; and compound 2 appears particularly promising for the severe phase, when BChE plays a more dominant role.
- MeSH
- Acetylcholinesterase metabolism chemistry MeSH
- Alzheimer Disease * drug therapy MeSH
- Butyrylcholinesterase metabolism chemistry MeSH
- Cholinesterase Inhibitors * chemistry pharmacology metabolism therapeutic use chemical synthesis MeSH
- Hydrazones * chemistry pharmacology MeSH
- Kinetics MeSH
- Humans MeSH
- Models, Molecular MeSH
- Drug Design * MeSH
- Semicarbazones * chemistry pharmacology metabolism MeSH
- Molecular Docking Simulation MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The polymerase acidic (PA) subunit of the influenza virus, an endonuclease of the RNA-dependent RNA polymerase, represents a viable target for anti-influenza therapies, as evidenced by the efficacy of the FDA-approved drug Xofluza. A characteristic feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions within the enzyme's catalytic site. Previously, our studies identified luteolin and its C-8-glucoside orientin as potent endonuclease inhibitors. This report details our subsequent investigation into the structural modifications of the phenyl moiety attached to the C-8 position of luteolin. The inhibitory potencies (IC50 values) quantified with AlphaScreen technology indicated that substituting the C-8 glucose moiety of orientin resulted in compounds with comparable inhibitory potency. From a series of eighteen compounds, acid 12 with 3-carboxylphenyl moiety at the C-8 position was the most potent inhibitor with nanomolar potency.
- MeSH
- Antiviral Agents * pharmacology chemical synthesis chemistry MeSH
- Endonucleases * antagonists & inhibitors metabolism MeSH
- Enzyme Inhibitors * pharmacology chemical synthesis chemistry MeSH
- Luteolin * pharmacology chemical synthesis chemistry MeSH
- Molecular Structure MeSH
- Drug Design * MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are two important members of the Tec family with crucial roles in immune system function. Deregulation in ITK and BTK activity is linked to several hematological malignancies, making them key targets for cancer immunotherapy. In this study, we synthesized a series of azaspirooxindolinone derivatives and evaluated their cytotoxic activity against ITK/BTK-negative and positive cancer cell lines, followed by enzymatic inhibition studies to assess the ITK/BTK kinase selectivity of two hit compounds. Several compounds demonstrated selective cytotoxicity against ITK- or BTK-expressing cells. Compound 3d exhibited high cytotoxicity in ITK-positive Jurkat (IC50 = 3.58 μM) and BTK-positive Ramos (IC50 = 3.06 μM) cells, while compound 3j showed strong cytotoxicity in Ramos (IC50 = 1.38 μM) and Jurkat (IC50 = 4.16 μM) cells. Compounds 3a and 3e were selectively cytotoxic in Jurkat cells (IC50 = 9.36 μM and 10.85 μM, respectively), while compounds 3f and 3g were highly cytotoxic in Ramos cells (IC50 = 1.82 μM and 1.42 μM, respectively). None of the active compounds exhibited cytotoxicity in non-cancer cell lines (IC50 > 50 μM), demonstrating their selectivity for malignant cells. Enzyme inhibition assay showed that 3d is a selective ITK inhibitor (IC50 = 0.91 μM) with no detectable BTK inhibition, aligning with its strong activity in ITK-positive cells. In contrast, compound 3j did not inhibit ITK or BTK enzymatically, suggesting an alternative mechanism of action. These findings highlight 3d as a promising ITK inhibitor and warrant further investigation to elucidate its mechanism of action.
- MeSH
- Protein Kinase Inhibitors * pharmacology chemical synthesis chemistry MeSH
- Humans MeSH
- Molecular Structure MeSH
- Cell Line, Tumor MeSH
- Oxindoles pharmacology chemistry chemical synthesis MeSH
- Cell Proliferation drug effects MeSH
- Agammaglobulinaemia Tyrosine Kinase * antagonists & inhibitors metabolism MeSH
- Antineoplastic Agents * pharmacology chemical synthesis chemistry MeSH
- Drug Design * MeSH
- Drug Screening Assays, Antitumor * MeSH
- Molecular Docking Simulation MeSH
- Spiro Compounds chemistry pharmacology chemical synthesis MeSH
- Protein-Tyrosine Kinases * antagonists & inhibitors metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower μM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 μM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.
- MeSH
- Antiviral Agents pharmacology chemistry chemical synthesis MeSH
- Bunyaviridae drug effects metabolism MeSH
- Endonucleases * metabolism antagonists & inhibitors MeSH
- Enzyme Inhibitors pharmacology chemistry chemical synthesis MeSH
- Isoindoles chemical synthesis pharmacology chemistry MeSH
- Humans MeSH
- Molecular Structure MeSH
- Drug Design * MeSH
- Molecular Docking Simulation * MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.
- MeSH
- Enzyme Inhibitors * pharmacology chemistry chemical synthesis MeSH
- Catalytic Domain MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Pentosyltransferases * antagonists & inhibitors metabolism MeSH
- Drug Design * MeSH
- Trypanosoma brucei brucei drug effects enzymology MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Organophosphorus compounds (OP) make up an important class of inhibitors, mostly employed as pesticides, even as chemical weapons. These toxic substances act through the inhibition of the acetylcholinesterase (AChE) enzyme, which results in elevated synaptic acetylcholine (ACh) levels, leading to serious adverse effects under the cholinergic syndrome. Many reactivators have been developed to combat the toxic effects of these AChE inhibitors. In this line, the oximes highlight because of their good reactivating power of cholinesterase enzymes. To date, no universal antidotes can reactivate AChE inhibited by any OP agent. This review summarizes the intoxication process by neurotoxic OP agents, along with the development of reactivators capable of reversing their effects, approaching aspects like the therapeutic and toxicological profile of these antidotes. Computational methods and conscious in vitro studies, capable of significantly predicting the toxicological profile of these drug candidates, might support the process of development of these reactivators before entering in vivo studies in animals, and then clinical trials. These approaches can assist in the design of safer and more effective molecules, reducing related cost and time for the process.
- MeSH
- Acetylcholinesterase chemistry MeSH
- Antidotes * pharmacology therapeutic use chemistry MeSH
- Cholinesterase Inhibitors toxicity MeSH
- Organophosphorus Compounds MeSH
- Oximes therapeutic use toxicity MeSH
- Cholinesterase Reactivators * therapeutic use toxicity MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Cardiovascular diseases, such as myocardial infarction, ischemic stroke, and pulmonary embolism, are the most common causes of disability and death worldwide. Blood clot hydrolysis by thrombolytic enzymes and thrombectomy are key clinical interventions. The most widely used thrombolytic enzyme is alteplase, which has been used in clinical practice since 1986. Another clinically used thrombolytic protein is tenecteplase, which has modified epitopes and engineered glycosylation sites, suggesting that carbohydrate modification in thrombolytic enzymes is a viable strategy for their improvement. This comprehensive review summarizes current knowledge on computational and experimental identification of glycosylation sites and glycan identity, together with methods used for their reengineering. Practical examples from previous studies focus on modification of glycosylations in thrombolytics, e.g., alteplase, tenecteplase, reteplase, urokinase, saruplase, and desmoteplase. Collected clinical data on these glycoproteins demonstrate the great potential of this engineering strategy. Outstanding combinatorics originating from multiple glycosylation sites and the vast variety of covalently attached glycan species can be addressed by directed evolution or rational design. Directed evolution pipelines would benefit from more efficient cell-free expression and high-throughput screening assays, while rational design must employ structure prediction by machine learning and in silico characterization by supercomputing. Perspectives on challenges and opportunities for improvement of thrombolytic enzymes by engineering and evolution of protein glycosylation are provided.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug resistance. New compounds with new mechanisms of action are therefore needed. We present the design, synthesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target - mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines. Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv). Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted benzamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is discussed.
- MeSH
- Aminopeptidases antagonists & inhibitors metabolism MeSH
- Antitubercular Agents MeSH
- Enzyme Inhibitors chemical synthesis chemistry pharmacology MeSH
- Microbial Sensitivity Tests MeSH
- Molecular Structure MeSH
- Mycobacterium tuberculosis drug effects enzymology MeSH
- Pyrazines chemical synthesis chemistry pharmacology MeSH
- Drug Design * MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Twenty-four novel compounds bearing tetrahydroacridine and N-propargyl moieties have been designed, synthesised, and evaluated in vitro for their anti-cholinesterase and anti-monoamine oxidase activities. Propargyltacrine 23 (IC50 = 21 nM) was the most potent acetylcholinesterase (AChE) inhibitor, compound 20 (IC50 = 78 nM) showed the best inhibitory human butyrylcholinesterase (hBChE) profile, and ligand 21 afforded equipotent and significant values on both ChEs (human AChE [hAChE]: IC50 = 0.095 ± 0.001 μM; hBChE: IC50 = 0.093 ± 0.003 μM). Regarding MAO inhibition, compounds 7, 15, and 25 demonstrated the highest inhibitory potential towards hMAO-B (IC50 = 163, 40, and 170 nM, respectively). In all, compounds 7, 15, 20, 21, 23, and 25 exhibiting the most balanced pharmacological profile, were submitted to permeability and cell viability tests. As a result, 7-phenoxy-N-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroacridin-9-amine hydrochloride (15) has been identified as a permeable agent that shows a balanced pharmacological profile [IC50 (hAChE) = 1.472 ± 0.024 μM; IC50 (hBChE) = 0.659 ± 0.077 μM; IC50 (hMAO-B) = 40.39 ± 5.98 nM], and consequently, as a new hit-ligand that deserves further investigation, in particular in vivo analyses, as the preliminary cell viability test results reported here suggest that this is a relatively safe therapeutic agent.
- MeSH
- Acetylcholinesterase metabolism MeSH
- Alzheimer Disease * drug therapy MeSH
- Amines MeSH
- Butyrylcholinesterase * metabolism MeSH
- Cholinesterase Inhibitors pharmacology therapeutic use MeSH
- Monoamine Oxidase Inhibitors pharmacology MeSH
- Humans MeSH
- Ligands MeSH
- Monoamine Oxidase MeSH
- Oxidoreductases MeSH
- Drug Design MeSH
- Tacrine therapeutic use MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.
