Activity-based probes form covalent bonds with active enzymes and can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. They have a reactive head group that covalently binds to the target, a tag that allows detection (e.g. a fluorophore) and a linker to connect both.^[1]

Matriptase-2 is a transmembrane, multi-domain serine protease with primary substrate specificity for arginine in P1 position, which plays a key role in the human iron homeostasis.^[2] Our design of activity-based probes for matriptase-2 is based on linker-connected bis-benzguanidines.^[3] The two benzguanidine units interact as arginine mimetics with the S1 and the upper part of the S3/S4 pocket, respectively, and direct the inhibitor to the active site of the target enzyme.^[2] An amino acid was introduced as a linker, which bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine.^[4] The resulting irreversible mode of action was demonstrated, leading to an enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase-2.

Herein, we present the preparation of coumarin-functionalized amino acids and the subsequent linear synthetic approach to coumarin-labeled bis-benzguanidines as activity-based probes for matriptase-2. Spectral properties and kinetic parameters for the reaction with matriptase-2 are reported.

References

1. Serim, S.; Haedke, U.; Verhelst, S. H. Chem. Med. Chem. 2012, 7, 1146-1159.
2. Stirnberg, M.; Gütschow, M. Curr. Pharm. Des. 2013, 19, 1052-1061.
3. Dosa, S.; Stirnberg, M.; Lülsdorff, V.; Häußler, D.; Maurer, E.; Gütschow, M. Bioorg. Med. Chem. 2012, 20, 6489-6505.
4. Scienczyk, M.; Oleksyszyn, J. Curr. Med. Chem. 2009, 16, 1673-1687.

Casein kinase 2 (CK2) is ubiquitous kinase protein emerging as a target for several human diseases including cancer. Several active CK2 inhibitors have been developed in the last few years; most of them have ATP-competitive type of inhibition, and only one inhibitor is in clinical trial as anticancer drug. Here we report on the synthesis of two derivatives of 2,6-diaryl-anthracene-9,10-dione, one of them, 2,6-di(furan-3-yl)anthracene-9,10-dione compound 3, turned out to be active towards CK2, and ATP competitive with an IC₅₀ value of 2.35 µM and a K_i value of 1.26 µM. Molecular modeling studies were performed using (MOE) to explain the binding affinity of compound 3 in comparison to emodin. These studies indicated that unlike emodin, compound 3 was not able to perform a hydrogen bond with Lys68, although the compound fits well in the active site of human CK2α, which explains the difference in the measured affinity between those two compounds.

The discovery of the physiological role of a great number of peptides (e.g. angiotensin II, neuropeptide Y, enkephalin, gonadotrophin-releasing hormone…) stimulated researchers towards design and synthesis of analogues. Since the last two decades, peptidomimetics have emerged as promising therapeutic agents such as goserelin, cetrorelix, and atazanavir. Structural modifications of the sequence of the native peptides can optimize their biological properties such as bioavailibilty, plasma half-life, resistance of metabolism and selectivity. Another advantage to develop peptidomimetics as drugs and/or probes can be the control of their conformation. A peptidomimetic with a restricted conformational flexibility can minimize binding to non-target receptors and then enhances the activity at the target receptor or transporter. Taking all together, peptidomimetics constitute a class of promising biomolecules to study ligand-protein interactions on different biological targets.

For many years, our researchers worked on multidrug resistance (MDR) to anticancer and anti-infectious agents. This phenomenon is often associated with over-expression of several proteins belonging to ABC transporters (e.g. ABCB1, ABCG2). Numerous molecules have shown activities on these transporters. Among them, we can list steroids, bivalent ligands, azaheterocycles and short linear hydrophobic peptides. For example, reversin 121, a dipeptide, showed high affinity and specificity for ABCB1.

Reversin 121 became the new starting point of our research. For 2005 we developed different series of (aza)peptidomimetic-type ligands of ABC transporters. First, we wish to expose on the synthesis work accomplished to reach our selection of molecules and secondly to present our biological data on ABCB1, and ABCG2.

Osteogenesis is a complex biological process that includes synthesis of an organic matrix composed mainly of type I collagen and mineralization of the organic matrix by deposition of hydroxyapatite crystals. Mutations in the PHEX gene, a gene encoding a peptidase (formerly PEX; Phosphate regulating gene with homologies to endopeptidases on the X chromosome), are responsible for X-linked hypophosphatemic rickets; a genetic disease that is characterized by undermineralization of the bone extracellular matrix. Several experimental observations support a role for PHEX in mineralization. Here we report on the design, synthesis, and in vitro biological activity of mercaptoacyl dipeptide-based inhibitors of PHEX. A parallel solid phase peptide synthesis approach was used for producing focused compound libraries resulting in single digit nanomolar PHEX inhibitors. Structure activity relationships studies revealed that the P1’ aspartic acid residue is critical and its deletion or modification lead to a large decrease in activity. The stereochemistry of the aspartic acid residue at P1’ is also important. Replacing L-aspartic acid with its D enantiomer led to about a seven fold loss of potency. We explored multiple sites of diversity around the central aspartic acid and these results are also reported. In assessing selectivity for PHEX versus NEP, all the derivatives tested were highly selective for PHEX. Such compounds may have potential usage in regulating bone mineralization and/or as osteogenic agents.

Sensors based on the molecular recognition of bio-molecules have already attracted intensive interest in many different fields including medical diagnostics and control, environmental monitoring, water treatment facilities, trace gas detection, analysis of biotechnological processes and quality control for food industries. Different surface sensitive techniques can be applied to detect these molecular interactions, depending on the nature of the sensor supports. Among those are optical sensors such as Surface Plasmon Resonance (SPR) or spectroscopy based-sensors. We propose to assess the utility of Fourier Transform Infrared (FTIR) spectroscopy in studying biomolecules attachment to inorganic surfaces in a variety of biosensing applications. We have designed a new generic device suitable for the investigation of ligand–receptor interactions based on successive grafting of a novel silanization reagent and a bifunctional molecular clip directly at the surface of an internal reflection element. These molecular constructions lead to activated transducer substrate (typically made of silicon or germanium) ready for the covalent binding of any bio-receptor molecules. Contrarily to SPR or quartz crystal microbalance (QCM) sensors, FTIR sensors provide useful spectroscopic information concerning the chemical nature of the interacting molecules, the amount of bound receptors and ligands, and even possible conformational transitions of the receptor during the interaction with the ligand can also be monitored. Currently, these informations are usually not accessible using standard sensors that are usually limited to measure physical modifications onto the surface. As the chemical structure of the interacting molecules is directly probed, FTIR-based sensors are de facto true label-free sensors. We will illustrate attachment of biomolecules to such organic surfaces through of various model systems commonly used in the biosensing field.

INTRODUCTION: Human adenovirus and herpes simplex virus cause infectious diseases of eyes, respiratory, enteric and urogenital tracts, of central and peripheral nervous system and are capable to persistence in latent form and are activated under the influence of endogenous and exogenous factors. A high spread of infections caused by herpes virus and adenovirus and their mixed infections are the problems of modern medicine. During mixed infections, an absence of viruses’ interaction as well as a mutual influence of viruses may originate. Search of drugs with a relatively high activity against the co-associated viruses by inhibiting their reproduction and transmission is an important task. Activity of antiviral drugs under condition of co-infection is not studied enough.
METHODS: Human adenovirus type 5 (HadV-5) and herpes simplex virus type 1 (HSV-1/US) were used as the model viruses. The reproduction of viruses in MDBK cells was studied using the cell-ELISA, flow cytometry and Western-blot. The levels of synthesis of the major proteins of adenoviruses and herpes viruses under conditions of the mixed infection were studied. The created model of mixed infection in the populations of MDBK cells was confirmed by the electron microscopy and immunofluorescence analysis.
The cytotoxic activity of the drugs was studied by the MTT-assay. The model of co-infection of MDBK cells was used to evaluate the efficiency of the antiviral agents of the broad action spectrum (ganciclovir, cidofovir and ribavirin) against HAdV-5 and HSV-1/US. The antiviral effects of the drugs were studied using the medical scheme of introduction of preparation (as a component of supporting medium). The analysis of the virus DNA synthesis in infected cells after treatment with the drugs was carried out by the RT-qPCR. The effectiveness of the preparations under mono- and mixed infections was studied by the cytomorphological method.
RESULTS: The simultaneous infection of the cells with two viruses resulted in a significant inhibition of the reproduction of herpes virus and in a less pronounced inhibition of the adenovirus reproduction.
Use of ribavirin in conditions of mono infection led to the inhibition of DNA replication of adenovirus by 41% and herpes virus by 29%. Ribavirin was ineffective against HАdV-5 and its activity against herpes virus remained unchanged under conditions of co-infection. The analysis of antiviral activity of ganciclovir on the models of mono infections showed the reduction of reproduction of HSV-1/US by 60% and HAdV-5 by 4%. Ganciclovir inhibited the reproduction of herpes virus by 61% during the co-infection. The effectiveness of the drug against adenovirus increased 20%. Under condition of the mixed infection the cidofovir activity against HAdV-5 and HSV-1/US decreased 3-fold and 79-fold, respectively, compared to the mono infections.
CONCLUSIONS: The changes of the nature of the pathological processes were found, as a result of the interference of the viruses and the differences of the drugs activities against the co-associated viruses in conditions of the mono and mixed infections of MDBK cells. Both the increase and inhibition of the drugs activities were detected that may lead to the formation of resistant strains of viruses.

In our continuous effort aiming at preparing novel heterocyclic scaffolds able to modulate the activity of kinases in signal transduction, thiazolo[5,4-f]quinazolines were particularly studied. This presentation describes a novel strategy for a convenient structure-activity-relationship study towards five serine/threonine kinases (CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3α/β) involved in Alzheimer’s disease.

The chemical highlight of this work was the use of Appel salt (4,5-dichloro-1,2,3-dithiazolium chloride) for the conception of 6-amino-2-cyanobenzo[d]thiazole-7-carboxylate derivatives as a versatile molecular platform from the 5-nitroanthranilic acid. Thus, introduction of various aliphatic, aromatic or amino substituents at position 8 was best achieved by one-pot DMFDMA-mediated cyclisation. Transformation of carbonitrile group into various chemical functions (e.g. imidate, ester, amidine...) allowed the efficient preparation of a library of novel thiazoloquinazoline derivatives. The first biological results have identified great and selective inhibition against DYRK1A and DYRK1B. The more active compounds are imidate derivatives exhibiting inhibitory activity in a subnanomolar range against DYRK1A.

            The neurotransmitter acetylcholine (ACh) exerts its effects on the central nervous system (CNS) through two distinct muscarinic mAChRs and nicotinic nAChRs receptors types: nAChRs belong to the superfamily of ligand-gated ion channels possessing a pentameric structure.¹ Because of their distribution and abundance in the CNS (in particular in the hippocampus and cortex), the a7 subtypes are potential diagnosis and therapeutic targets for brain disorders that involve these cerebral regions. In this field, α7 nAChR agonists were identified and allowed the design of novel therapeutic agents for Alzheimer Disease (AD). Having in hand a human compatible [¹⁸F]-labeled positron emission tomography (PET) tracer to realize the early diagnostic or to validate the efficiency of therapies in clinical trials for AD is indubitably crucial.

            In this aim, based on our expertise in heterocyclic bio-mimetic development,² we also envisioned to design novel α7 nAChR ligands and their transformation into a [¹⁸F] PET tracer. We synthesized a library of potent α7 nAChR ligands containing a quinuclidine, a tropane or a 8H-quinolizine moiety. We present herein chemistry,³ SAR studies, molecular modelling docking studies which confirmed the binding mode of the developed ligands, in vitro efficiency (SAR), radiolabeling and in vivo results in rats.

1. J. Wu, M. Ishikawa, J. Zhang and K. Hashimoto Int. J. Alzheimers Dis., 2010, 2010, 11; F. Dajas-Bailador and S. Wonnacott., Trends Pharm. Sci., 2004, 25, 317. C. Gotti, M. Zoli and F. Clementi, Trends Pharm. Sci., 2006, 27, 482; D. Paterson and A. Nordberg, Prog. neurobiol., 2000, 61, 75.
2. C. Neagoie, E. Vedrenne, F. Buron, J. Y Merour, S. Rosca, S. Bourg, O. Lozach, L. Meijer, B. Baldeyrou, A. Lansiaux and S. Routier, Eur. J. Med. Chem. 2012, 49, 379; R. Boulahjar ; A. Ouach, M. Chiurato, S. Bourg, M. Ravache, R. Le Guével, S. Marionneau-Lambot, T. Oullier, O. Lozach, L. Meijer, C. Guguen-Guillouzo, S. Lazar, M. Akssira, Y. Troin, G. Guillaumet and S. Routier, J. Med. Chem., 2012, 55, 9589;
3. Routier, S. ; Suzenet, F. ; Pin, F. ; Chalon, S. ; Vercouillie, J. ; Guilloteau, D. WO 2012143526 ; F. Pin, J.Vercouillie, A. Ouach, S. Mavel, Z. Gulhan, G. Chicheri, C. Jarry, S. Massip, J.-B. Deloye, D. Guilloteau, F. Suzenet, S. Chalon S. Routier, Euj J. Med; Chem. . 2014, 83, 214-224.

Research Support: ANR Malz MInAlpha 7,the Labex Iron (ANR-11-LABX-18-01), the Région Centre (IMAD), the FEDER and Cyclopharma Laboratories.

Keywords:

Aims of the study were to prepare and investigate the dissolution and floatability profiles of Nizatidine and Piracetam effervescent floating tablets and to study the effect of Xanthan Gum, Eudragit-RS PO or Lubritose SD on tablet compression properties with or without granulation of the powder admixtures. Sodium bicarbonate was used to release CO₂ when tablets come in contact with the acidic medium. Tablets without drugs were characterised for their floatability properties in simulated gastric fluid (SGF) without enzymes at 37°C. The successful formulations regarding floatability were incorporated with Nizatidine (50mg/tablet) or Piracetam (30mg/tablet). The powder admixtures were characterised for flow properties and tablets containing drugs were evaluated via British Pharmacopeia quality control tests.

All batches with Nizatidine that contain Xanthan Gum alone or in combination with Eudragit-RS PO showed good flow and compaction properties and also yielded significant (p<0.05) swelling and floating results. However, Piracetam batches prepared with Lubritose SD showed poor compaction, therefore granulation of the powders was applied to enhance floating tablet properties such as friability, floatability and ustainability of the drug release for more than 6 hours.

In conclusion, Xanthan Gum and Eudragit-RS PO (used with Nizatidine) and Lubritose SD (applied with Piracetam) could be promising excipients to formulate floating tablets.

The proteolytic cleavage is the universal mechanism of specific proteins activation. The activation of proteolysis plays an important role in the pathogenesis of many diseases including viral infections. The results of our research and the scientific data have made it possible to formulate the concept of formation of a “vicious circle”: virus activates the proteolytic systems, which in turn assists the development, generalization, and aggravation of the infectious process at the expense of influence on the etiological and pathogenetical factors. Inhibitors of proteolysis (IP) may prevent the formation or destroy this “vicious circle”. Usually IP E-aminocaproic acid (name of medicine “Aminocaproic acid” - ACA) is used for    hemostasis when fibrinolysis contributes to bleeding. ACA is a low toxic drug. The intravenous and oral LD₅₀ of ACA are 3.0 and 12.0 g/kg respectively in mice. ACA prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into sensitive cells. It brings down proteolytic cleavage of HA precursor to HA-1 and HA-2 and reduces the infectious virus harvest. It has been shown that, in vitro, ACA exhibits high levels of anti-influenza efficacy on subtypes H1N1; H2N2; H3N2 of human, H5N3 and H7N3 avian influenza A viruses and on influenza B viruses. ACA promotes the intensification of specific antibodies production and cell immunity, prevents vessels permeability and hemorrhagic phenomena and decreases the destruction of bronchi epithelium. Mice treated by ACA after primary infection were more protected to re-infection. ACA decreased the virus reproduction in lungs and also enhanced  the humoral immune response when used  in the prevention and treatment of influenza. Farmaceutic Center and Ukrainian Ministry of Public Health, on the basis of our experimental research and clinical trials, have allowed using ACA as antiviral for prevention and treatment of influenza and other acute respiratory viral infections (ARVIs) in children and adults. The results of including ACA in the therapeutic complex for treatment of influenza and other ARVIs in children were: decrease of duration of intoxication symptoms on 2-3 days, of fever on 1.5-3 days, of catarrhal symptoms on 1.5-2 days. Number of complications was reduced to 17%. Prophylactic efficacy of ACA was also established. With the scope to study the ACA prophylactic activity in organized collectives, we have prescribed it per-orally in 2.0 g dose four times a day during a week. The monitoring was performed in two independent collectives (923 young adults males aged 18-19 years old) during acute respiratory diseases appraisal. As the reference, 4 groups were taken (2 from each collective) but without  ACA application. The obtained results show: compare to the high morbidity rates in the reference group of the first tested collective in the basic group preventively treated with ACA, number of ARVIs has decreased in two-fold. At the same time, compare to the morbidity rate growth of ARVIs (by 15-27%), quinsy (by 14-21%) and pneumonia (by 6-7%) in the reference groups of the second tested collective, of the main examined group treated with ACA, pneumonia morbidity rate has decreased up to five-fold, while the ARVIs and quinsy levels were stabilized. The smallest number of cases of these infections in both teams surveyed was recorded in the period of ACA application. The obtained results allow to recommend the use of ACA for the efficient prophylaxis of ARVIs, quinsy and pneumonia in the organized collectives in the period of increased incidences of these infections. It is known that use of agents with different mechanisms of antiviral actions may have as result synergistic effects. Our studies of anti-influenza activity in vitro and in vivo show that the results of combined action of ACA with Tamiflu are synergistic antiviral effects. We have studied the influence of ACA on the growth of S.aureus. Strains  АТСС 25923, 2781 and Kunda of Staphylococcus aureus were  used.  It was stated that ACA taken in final concentration of 15, 10 and 7.5%, has hindered the growth of S.aureusАТСС 25923 totally. It is known that the IP increases the sensitivity of some microorganisms to antibiotics. We have shown that  ACA inhibits the reproduction of various strains of S.aureus that have different sensitivity to antibiotics. ACA can strengthen the antimicrobial action of antibiotics against the studied strains of Staphylococcus aureus. So use of ACA for treatment of influenza and other ARVIs simultaneously prevents bacterial complications.

 Conclusion E-aminocaproic acid is an effective remedy for prevention and treatment of influenza  and  acute respiratory viral infections in children and adults and  simultaneously for prevention of bacterial complications. The combination of proteolytic inhibitor E-Aminocaproic acid with neuraminidase inhibitor Tamiflu constitutes a promising perspective in anti-influenza protection and therapy.