Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence.Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy –performed in a large and diverse chemolibrary– complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening.Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12–20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7–45 μM).Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known “garbage in, garbage out” machine learning principle

Design and synthesis of bioactive heterocyclic compounds on solid and solution phase: an application to natural products

Τhe present Ph.D.Τhesis involves the development of novel and versatile synthetic approaches to heterocyclic systems containing the β-dίcarbοnyl" and β,β’-trίcarbοnyl moiety, or similar heterocyclic structures. Most of synthetic targets were synthesized in solution but a considerable part of this PhD thesis is connected with the synthesis of these compounds on solid support. The core structures of these heterocyclic compounds are common features shared by numerous natural products isolated from marine and terrestrial microorganisms. The first synthetic protocol connected with the synthesis of five-membered oxygen heterocycles such as 3-subsituted and 5,5-disubsituted tetronic acids, 3-subsituted-5-arylidene tetronic acids and natural products Pulvinones, 3-subsituted-2-aminofuran-4-ones, using cyclic anhydrides of α-hydroxy acids as optical active acylating agents. As a continuation of our studies, we present the synthesis of 3-mono and 3,3-disubstituted 5-carboxymethyltetramic acids. The developed methodology utilizes N-acetyl aspartic acid anhydride as a chiral synthon, which serves as an stereoselective acylating agent for carbanions such as β-ketoesters and substituted acetates. In order to synthesize N-methyl 3-substituted and 3,3-disubstituted tetramic acids and N-methyl 3-substituted-2-aminopyrrolin-4-ones we have developed a new synthetic protocol involving the reaction of an appropriate N-carboxy-anhydride of N-methyl-amino acid with suitably functionalized carbanions. The N-H 3,3-disubstituted tetramic acids analoques have been synthesized by the use of N-acetyl N-carboxy-anhydrides. We document the development of a novel solid-phase traceless synthesis of functionalized five membered nitrogen heterocycles, namely 3-substituted-2,4-pyrrolinediones (tetramic acids) and 3-substituted-2-amino-4-pyrrolinones using a carbamate linker. Next we turned to the synthesis of 3-substituted-4-hydroxy-2-quinolinones. Our methodology involves the attachment of substituted acetic acids to the Wang resin. Τheir carbanions react with 2-ethoxybenzoxazinones gives the C-acylation resin bounds intermediates which cleaved to the desired compounds. Finally we developed a solid phase synthetic route to 3-alkoxycarbony-4-hydroxy-coumarins, 3-alkoxycarbony-4-hydroxyquinolin-4-ones and 3-cyano-2-aminochromones. This methodology involves the attachment of the corresponding active N-hydrohysuccinimide esters of substituted salicylic and anthranilic acids to the 2-chlorotrityl resin, followed by a reaction using β-ketoester carbanions in order to give the target molecules after the cleavage from the resin.Η παρούσα διδακτορική διατριβή έχει ως αντικείμενο την σύνθεση βιοδραστικών β-δικαρβονυλικών και β,β’-τρικαρβονυλικών ετεροκυκλικών ενώσεων, και παρόμοιων δομών. Οι περισσότερες ομάδες ενώσεων συντέθηκαν σε υγρή φάση, επιπλέον όμως ένα σημαντικό κομμάτι της διδακτορικής διατριβής αποτελεί και η σύνθεση ετεροκυκλικών ενώσεων σε στερεά φάση. Ο βασικός ετεροκυκλικός δακτύλιος των ενώσεων αυτών αποτελεί δομική μονάδα πολλών φυσικών προϊόντων τα οποία απομονώθηκαν από θαλάσσιους και χερσαίους μικροοργανισμούς. Συγκεκριμένα, η πρώτη μεθοδολογία η οποία αναπτύχθηκε ξεκίνησε με τη σύνθεση ετεροκυκλικών ενώσεων του οξυγόνου πενταμελούς δακτυλίου όπως 3-υποκατεστημένων και 5,5-δισυποκατεστημένων τετρονικών οξέων, 3-υποκατεστημένων-5-αρυλιδενοτετρονικών οξέων και των φυσικών προϊόντων Pulvinones και 3-υποκατεστημένων 2-αμινοφουραν-4-ονών, η οποία βασίζεται στη χρήση των κυκλικών ανυδριτών των α-υδροξυοξέων ως οπτικώς ενεργών ακυλιωτικών ενώσεων. Στη συνέχεια μια νέα συνθετική πρόταση για τη σύνθεση 3-υποκατεστημένων και 3,3-διυποκατεστημένων 5-καρβοξυμέθυλο τετραμικών οξέων με πρώτη ύλη τον Ν-ακετυλο ανυδρίτη του ασπαρτικού οξέος ο οποίος ακυλιώνει καρβανιόντα β-κετοεστέρων και υποκατεστημένων οξικών εστέρων έτσι ώστε να καταλήξουμε προς τα αντίστοιχα οπτικώς ενεργά τετραμικά οξέα. Στη προσπάθεια να συνθέσουμε Ν-μεθυλο 3-υποκατεστημένα και 3,3-δισυποκατεστημένα τετραμικά οξέα και 3-υποκατεστημένες 2-αμινοπυρρολιν-4-όνες έγινε χρηση των Ν-μεθυλο-καρβοξυανυδριτών και κατάλληλων καρβανιόντων εστέρων. Ν-Η 3,3-δισυποκατεστημένα τετραμικά οξέα συντέθηκαν από τους αντίστοιχους Ν-ακετυλο-καρβοξυανυδρίτες. Η σύνθεση 3-υποκατεστημένων τετραμικών οξέων και 2-αμινοπυρρολιν-4-ονών πραγματοποιήθηκε σε στερεά φάση μέσω ρητίνης ενεργού p-νιτροφαινυλεστερα του ανθρακικού οξέος σε ρητίνη Wang. Ανάλογες διαδικασίες σε στερεά φάση έγιναν με κατεύθυνση την σύνθεση 3-υποκατεστημένων 4-υδροξυ-κινολιν-2-ονών, μέσω αντιδράσεων οξικών εστέρων προσδεμένους σε ρητίνη Wang και υποκατεστημένων 2- αιθοξυβενζοξαζινονών. Τα καρβανιόντα των εστέρων αυτών ακυλιώνονται από τις βενζοξαζινόνες αποδίδοντας τα ενδιάμεσα C-ακυλιώσεως τα οποία με κυκλοποίηση και αποκοπή από τη ρητίνη οδηγουν στα επιθυμητά προϊόντα. Τέλος αναπτύξαμε συνθετικό μοντέλο σε στερεά φάση για τη σύνθεση 3-υποκατεστημένων 4-υδροξυ-κουμαρινών και 4-υδροξυ-κινολιν-2-ονών όπως επίσης και 3-κυανο-2-αμινοχρωμονών. Το μοντέλο αυτό περιλαμβάνει τη πρόσδεση Ν-υδροξυ­ηλεκτριμιδοεστέρων υποκατεστημένων σαλικυλικών και ανθρανιλικών οξέων σε 2-χλωροτρίτυλο ρητίνη. Οι αντιδράσεις τους με κατάλληλες ενώσεις ενεργού μεθυλενίου, οδήγησαν στα αντίστοιχα «ενδιάμεσα» όπου με αποκοπή από τη ρητίνη και ταυτόχρονη κυκλοποίηση κατέληξε στη σύνθεση των επιθυμητών προϊόντων

Solid-Phase Synthesis of Optically Active Substituted 2 Aminofuranones Using an Activated Carbonate Linker

An efficient three-step solid-phase synthesis of diverse 3,5-disubstituted-2-aminofuranones has been developed. α-Hydroxy acids loaded on a nitrophenyl carbonate derivative of Wang resin are used as acylating agents for the C-acylation of active methylene compounds and the resulting intermediates provided, through a cyclative cleavage reaction, the desired product

Synthesis and Antifouling Activity Evaluation of Analogs of Bromosphaerol, a Brominated Diterpene Isolated from the Red Alga Sphaerococcus coronopifolius

Marine biofouling is an epibiotic biological process that affects almost any kind of submerged surface, causing globally significant economic problems mainly for the shipping industry and aquaculture companies, and its prevention so far has been associated with adverse environmental effects for non-target organisms. Previously, we have identified bromosphaerol (1), a brominated diterpene isolated from the red alga Sphaerococcus coronopifolius, as a promising agent with significant antifouling activity, exerting strong anti-settlement activity against larvae of Amphibalanus (Balanus) amphitrite and very low toxicity. The significant antifouling activity and low toxicity of bromosphaerol (1) motivated us to explore its chemistry, aiming to optimize its antifouling potential through the preparation of a number of analogs. Following different synthetic routes, we successfully synthesized 15 structural analogs (2–16) of bromosphaerol (1), decorated with different functional groups. The anti-settlement activity (EC50) and the degree of toxicity (LC50) of the bromosphaerol derivatives were evaluated using cyprids and nauplii of the cirriped crustacean A. amphitrite as a model organism. Derivatives 2, 4, and 6–16 showed diverse levels of antifouling activity. Among them, compounds 9 and 13 can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism

Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity

Neurotrophins are growth factors that exert important neuroprotective effects by preventing neuronal death and synaptic loss. Nerve Growth Factor (NGF) acts through the activation of its high-affinity, pro-survival TrkA and low-affinity, pro-apoptotic p75NTR receptors. NGF has been shown to slow or prevent neurodegenerative signals in Alzheimer’s Disease (AD) progression. However, its low bioavailability and its blood–brain-barrier impermeability limit the use of NGF as a potential therapeutic agent against AD. Based on our previous findings on synthetic dehydroepiandrosterone derivatives, we identified a novel NGF mimetic, named ENT-A013, which selectively activates TrkA and exerts neuroprotective, anti-amyloid-β actions. We now report the chemical synthesis, in silico modelling, metabolic stability, CYP-mediated reaction phenotyping and biological characterization of ENT-A013 under physiological and neurodegenerative conditions. We show that ENT-A013 selectively activates the TrkA receptor and its downstream kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death. Moreover, ENT-A013 promotes survival of primary Dorsal Root Ganglion (DRG) neurons upon NGF withdrawal and protects hippocampal neurons against Amyloid β-induced apoptosis and synaptic loss. Furthermore, this neurotrophin mimetic partially restores LTP impairment. In conclusion, ENT-A013 represents a promising new lead molecule for developing therapeutics against neurodegenerative disorders, such as Alzheimer’s Disease, selectively targeting TrkA-mediated pro-survival signals

Data_Sheet_1_A quest for the stereo-electronic requirements for selective agonism for the neurotrophin receptors TrkA and TrkB in 17-spirocyclic-dehydroepiandrosterone derivatives.docx

IntroductionThe neurotrophin system plays a pivotal role in the development, morphology, and survival of the nervous system, and its dysregulation has been manifested in numerous neurodegenerative and neuroinflammatory diseases. Neurotrophins NGF and BDNF are major growth factors that prevent neuronal death and synaptic loss through binding with high affinity to their specific tropomyosin-related kinase receptors namely, TrkA and TrkB, respectively. The poor pharmacokinetic properties prohibit the use of neurotrophins as therapeutic agents. Our group has previously synthesized BNN27, a prototype small molecule based on dehydroepiandrosterone, mimicking NGF through the activation of the TrkA receptor.MethodsTo obtain a better understanding of the stereo-electronic requirements for selective activation of TrkA and TrkB receptors, 27 new dehydroepiandrosterone derivatives bearing a C17-spiro-dihydropyran or cyclobutyl moiety were synthesized. The new compounds were evaluated for their ability (a) to selectively activate the TrkA receptor and its downstream signaling kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death, and (b) to induce phosphorylation of TrkB and to promote cell survival under serum deprivation conditions in NIH3T3 cells stable transfected with the TrkB receptor and primary cortical astrocytes. In addition the metabolic stability and CYP-mediated reaction was assessed.ResultsAmong the novel derivatives, six were able to selectively protect PC12 cells through interaction with the TrkA receptor and five more to selectively protect TrkB-expressing cells via interaction with the TrkB receptor. In particular, compound ENT-A025 strongly induces TrkA and Erk1/2 phosphorylation, comparable to NGF, and can protect PC12 cells against serum deprivation-induced cell death. Furthermore, ENT-A065, ENT-A066, ENT-A068, ENT-A069, and ENT-A070 showed promising pro-survival effects in the PC12 cell line. Concerning TrkB agonists, ENT-A009 and ENT-A055 were able to induce phosphorylation of TrkB and reduce cell death levels in NIH3T3-TrkB cells. In addition, ENT-A076, ENT-A087, and ENT-A088 possessed antiapoptotic activity in NIH-3T3-TrkB cells exclusively mediated through the TrkB receptor. The metabolic stability and CYP-mediated reaction phenotyping of the potent analogs did not reveal any major liabilities.DiscussionWe have identified small molecule selective agonists of TrkA and TrkB receptors as promising lead neurotrophin mimetics for the development of potential therapeutics against neurodegenerative conditions.</p

Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

International audienceBACKGROUND:The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate.METHODOLOGY/PRINCIPAL FINDING:A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds.CONCLUSIONS/SIGNIFICANCE:Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors

Structure of compounds affecting tritryp trypanothione synthetase activity.

<p><b>AI (P)</b>, 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives, paullones derivatives, (FS-554 and MOL2008), five APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives (ZEA10, ZEA35, ZEA40, ZEA41 and ZVR159), eight BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives (EAP1-47, EAP1-63, APC1-67, APC1-87, APC1-89, APC1-99, APC1-101 and APC1-111), seven BBHPP, 1-(benzo[<i>d</i>]thiazol-2-yl)-4-benzoyl-3-hydroxy-5-phenyl-1<i>H</i>-pyrrol-2(5<i>H</i>)-one derivatives (AD81, AD84, ADMRC158, ADKPN160, ADKPN161, ADKPN164 and ADKPN165), three BZ, benzofuroxan derivatives (J18, J20 and J31) and one PD, 1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione derivatives [(<i>Z</i>)-8-(2-(2,4-dihydroxybenzylidene)hydrazinyl)-7-(2-hydroxy-3-phenoxy propyl)-1,3-dimethyl-1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione, TC227]. iPr, tBu, OBn, Mo and Ph, correspond to an isopropyl, tert-butyl, O-benzyl, 4 -morpholinyl and phenyl substitution, respectively.</p

Trypanothione dependent redox metabolism.

<p>The chemical structure of trypanothione (<i>N</i>¹,<i>N</i>⁸-bis(glutathionyl)spermidine; T(SH)₂) is depicted at the center. Synthesis: trypanothione synthetase catalyzes the ligation of two molecules of gluthatione to one of spermidine using the energy provided by two ATP molecules. Regeneration: trypanothione reductase maintains trypanothione in the reduced state at expenses of NADPH, which can be supplied by the oxidative phase of the pentose phosphate pathway <i>via</i> glucose 6-phosphate dehydrogenase. Utilization: reduced trypanothione is involved in multiple functions such as the detoxification of xenobiotics, cell proliferation, defense against oxidants and protein thiol-redox homeostasis. The multipurpose oxidoreductase tryparedoxin plays an important role catalyzing electron transfer from T(SH)₂ to different molecular targets (e.g. peroxidases, ribonucleotide reductase and protein disulfides). G6P: glucose-6-phosphate, 6PGL: 6-phosphogluconolactone, T(SH)₂: reduced trypanothione, TS_2[ox]: oxidized trypanothione, NDPs: nucleosides diphosphate, dNDP: deoxinucleosides diphosphate, E^-: electrophilic species, TS-E: trypanothione-electrophile adduct, ROOH: hydroperoxide, ONOOH: peroxynitrite, NO₂^-: nitrite.</p