Molecular glue degraders: exciting opportunities for novel drug discovery.

INTRODUCTION: Molecular Glue Degraders (MGDs) is a concept that refers to a class of compounds that facilitate the interaction between two proteins or molecules within a cell. These compounds act as bridge that enhances specific Protein-Protein Interactions (PPIs). Over the past decade, this technology has gained attention as a potential strategy to target proteins that were traditionally considered undruggable using small molecules. AREAS COVERED: This review presents the concept of cellular homeostasis and the balance between protein synthesis and protein degradation. The concept of protein degradation is concerned with molecular glues, which form part of the broader field of Targeted Protein Degradation (TPD). Next, pharmacochemical strategies for the rational design of MGDs are detailed and illustrated by examples of Ligand-Based (LBDD), Structure-Based (SBDD) and Fragment-Based Drug Design (FBDD). EXPERT OPINION: Expanding the scope of what can be effectively targeted in the development of treatments for diseases that are incurable or resistant to conventional therapies offers new therapeutic options. The treatment of microbial infections and neurodegenerative diseases is a major societal challenge, and the discovery of MGDs appears to be a promising avenue. Combining different approaches to discover and exploit a variety of innovative therapeutic agents will create opportunities to treat diseases that are still incurable.

UHPLC-MS/MS method for the quantification of ultra-traces of irinotecan and its metabolites in red blood cells and plasma to detect caregivers' contamination.

The occupational exposure of caregivers to antineoplastic agents has been demonstrated since 1979. Since the early 1990s, numerous studies from several countries have demonstrated the contamination of care facilities by antineoplastic drugs. As it is easier to sample, most contamination measurements in workers are carried out in urine sample. The distribution and elimination half-lives of irinotecan suggest that blood can be considered as better than urine for the biomonitoring of a potential contamination of healthcare workers. We describe here the development and the validation of a UHPLC-MS/MS method to simultaneously quantify irinotecan, and two of its main metabolites, APC and SN-38, at ultra-trace levels in plasma and red blood cells (RBC). This method has been applied to blood samples collected from several healthcare services in a French comprehensive cancer center. The results demonstrate that the method is sensitive enough to identify a contamination of healthcare workers by irinotecan and SN-38 at very low concentrations. Moreover, the results show that analysis of RBC is of great interest and complementary to that of serum.

A Biomimetic Multiparametric Assay to Characterise Anti-Amyloid Drugs.

Alzheimer's disease (AD) is the most widespread form of senile dementia worldwide and represents a leading socioeconomic problem in healthcare. Although it is widely debated, the aggregation of the amyloid ß peptide (Aß) is linked to the onset and progression of this neurodegenerative disease. Molecules capable of interfering with specific steps in the fibrillation process remain of pharmacological interest. To identify such compounds, we have set up a small molecule screening process combining multiple experimental methods (UV and florescence spectrometry, ITC, and ATR-FTIR) to identify and characterise potential modulators of Aß1-42 fibrillation through the description of the biochemical interactions (molecule-membrane Aß peptide). Three known modulators, namely bexarotene, Chicago sky blue and indomethacin, have been evaluated through this process, and their modulation mechanism in the presence of a biomembrane has been described. Such a well-adapted physico-chemical approach to drug discovery proves to be an undeniable asset for the rapid characterisation of compounds of therapeutic interest for Alzheimer's disease. This strategy could be adapted and transposed to search for modulators of other amyloids such as tau protein.

Novel thienopyrimidones targeting hepatic and erythrocytic stages of Plasmodium parasites with increased microsomal stability.

Based on the structure of a previously identified hit, Gamhepathiopine 1, which showed promising antiplasmodial activity, but poor microsomal stability, several strategies were investigated to improve the metabolic stability of the compounds. This included the introduction of fluorine or deuterium atoms, as well as carbocyclic groups. Among the new compounds, the 2-aminocyclobutyl derivative 5g demonstrated enhanced microsomal stability compared to compound 1, while retaining antiplasmodial activity against erythrocytic and hepatic stages of Plasmodium, without significant cytotoxicity against primary hepatocytes.

Design, synthesis and biological characterization of novel activators of the TrkB neurotrophin receptor.

Numerous studies have been published about the implication of the neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, Multiple Sclerosis and motor neuron disease. BDNF activates the TrkB receptor with high potency and specificity, promoting neuronal survival, differentiation and synaptic plasticity. Based on the main structural characteristics of LM22A-4, a previously published small molecule that acts as activator of the TrkB receptor, we have designed and synthesized a small data set of compounds. The lead idea for the design of the new compounds was to modify the third position of the LM22A-4, by introducing different substitutions in order to obtain compounds which will have not only better physicochemical properties but selective activity as well. ADME and toxicity profiles of molecules have been evaluated as well as their biological properties through the TrkB receptor and affinity to promote neurite differentiation.

Improving Aqueous Solubility and In Vitro Pharmacokinetic Properties of the 3-Nitroimidazo[1,2-a]pyridine Antileishmanial Pharmacophore.

An antileishmanial structure−activity relationship (SAR) study focused on positions 2 and 8 of the imidazo[1,2-a]pyridine ring was conducted through the synthesis of 22 new derivatives. After being screened on the promatigote and axenic amastigote stages of Leishmania donovani and L. infantum, the best compounds were tested against the intracellular amastigote stage of L. infantum and evaluated regarding their in vitro physicochemical and pharmacokinetic properties, leading to the discovery of a new antileishmanial6-chloro-3-nitro-8-(pyridin-4-yl)-2-[(3,3,3-trifluoropropylsulfonyl)methyl]imidazo[1,2-a]pyridine hit. It displayed low cytotoxicities on both HepG2 and THP1 cell lines (CC50 > 100 µM) associated with a good activity against the intracellular amastigote stage of L. infantum (EC50 = 3.7 µM versus 0.4 and 15.9 µM for miltefosine and fexinidazole, used as antileishmanial drug references). Moreover, in comparison with previously reported derivatives in the studied series, this compound displayed greatly improved aqueous solubility, good mouse microsomal stability (T1/2 > 40 min) and high gastrointestinal permeability in a PAMPA model, making it an ideal candidate for further in vivo studies on an infectious mouse model.

Synthesis of antiplasmodial 2-aminothieno[3,2-d]pyrimidin-4(3H)-one analogues using the scaffold hopping strategy.

Gamhepathiopine (also known as M1), is a multi-stage acting antiplasmodial 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one hydrochloride that was first described in 2015. The development of this compound is limited by poor microsomal stability, insufficient aqueous solubility and low intestinal permeability. In order to obtain new optimized derivatives, we conducted a scaffold hopping strategy from compound M1, resulting in the synthesis of 20 new compounds belonging to six chemical series. All the compounds were tested on the K1 multi-resistant strain of Plasmodium falciparum and the human HepG2 cell-line, to evaluate their antiplasmodial activity and their cytotoxicity. Analogues' biological results also highlighted the mandatory presence of a heteroatom at position 5 of the thieno[3,2-d]pyrimidin-4(3H)-one moeity for the antiplasmodial activity. However, modifications at position 7 were detrimental for the antiplasmodial activity. We identified furane bioisostere 3j as a promising candidate, showing good blood stage antiplasmodial activity, better water solubility and highly improved intestinal permeability in the PAMPA assay.

ß-Amyloid peptide interactions with biomimetic membranes: A multiparametric characterization.

Alzheimer's disease is the most common form of senile dementia in the world, and amyloid ß peptide1-42 (Aß1-42) is one of its two principal biological hallmarks. While interactome concept was getting forward the scientific community, we proposed that the study of the molecular interactions of amyloid ß peptide with the biological membranes will allow to highlight underlying mechanisms responsive of AD. We have developed two simple liposomal formulations (phosphatidylcholine, cholesterol, phosphatidylglycerol) mimicking neuronal cell membrane (composition, charge, curvature radius). Interactions with Aß1-42 and mutant oG37C, a stable oligomeric form of the peptide, were characterized according to a simple multiparametric procedure based on ThT fluorescence, liposome leakage assay, ATR-FTIR spectroscopy. Kinetic aggregation, membrane damage and peptide conformation provided our first methodologic bases to develop an original model to describe interactions of Aß peptide and lipids.

Cyclic Poly(α-peptoid)s by Lithium bis(trimethylsilyl)amide (LiHMDS)-Mediated Ring-Expansion Polymerization: Simple Access to Bioactive Backbones.

Cyclic polymers display unique physicochemical and biological properties. However, their development is often limited by their challenging preparation. In this work, we present a simple route to cyclic poly(α-peptoids) from N-alkylated-N-carboxyanhydrides (NNCA) using LiHMDS promoted ring-expansion polymerization (REP) in DMF. This new method allows the unprecedented use of lysine-like monomers in REP to design bioactive macrocycles bearing pharmaceutical potential against Clostridioides difficile, a bacterium responsible for nosocomial infections.

Pleiotropic prodrugs: Design of a dual butyrylcholinesterase inhibitor and 5-HT6 receptor antagonist with therapeutic interest in Alzheimer's disease.

Beside acetylcholinesterase, butyrylcholinesterase could be considered as a putative target of interest for the symptomatic treatment of Alzheimer's disease (AD). As a result of complexity of AD, no molecule has been approved since 2002. Idalopirdine, a 5-HT6 receptors antagonist, did not show its effectiveness in clinical trial despite its evaluation as adjunct to cholinesterase inhibitors. Pleiotropic molecules, known as multitarget directed ligands (MTDLs) are currently developed to tackle the multifactorial origin of AD. In this context, we have developed a pleiotropic carbamate 7, that behaves as a covalent inhibitor of BuChE (IC50 = 0.97 µM). The latter will deliver after hydrolysis, compound 6, a potent 5-HT6 receptors antagonist (Ki = 11.4 nM) related to idalopirdine. In silico and in vitro evaluation proving our concept were performed completed with first in vivo results that demonstrate great promise in restoring working memory.

A functional m6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development.

N6 -methyladenosine (m6 A) is a prevalent epitranscriptomic mark in eukaryotic RNA, with crucial roles for mammalian and ecdysozoan development. Indeed, m6 A-RNA and the related protein machinery are important for splicing, translation, maternal-to-zygotic transition and cell differentiation. However, to date, the presence of an m6 A-RNA pathway remains unknown in more distant animals, questioning the evolution and significance of the epitranscriptomic regulation. Therefore, we investigated the m6 A-RNA pathway in the oyster Crassostrea gigas, a lophotrochozoan model whose development was demonstrated under strong epigenetic influence. Using mass spectrometry and dot blot assays, we demonstrated that m6 A-RNA is actually present in the oyster and displays variations throughout early oyster development, with the lowest levels at the end of cleavage. In parallel, by in silico analyses, we were able to characterize at the molecular level a complete and conserved putative m6 A machinery. The expression levels of the identified putative m6 A writers, erasers and readers were strongly regulated across oyster development. Finally, RNA pull-down coupled to LC-MS/MS allowed us to prove the actual presence of readers able to bind m6 A-RNA and exhibiting specific developmental patterns. Altogether, our results demonstrate the conservation of a complete m6 A-RNA pathway in the oyster and strongly suggest its implication in early developmental processes including MZT. This first demonstration and characterization of an epitranscriptomic regulation in a lophotrochozoan model, potentially involved in the embryogenesis, bring new insights into our understanding of developmental epigenetic processes and their evolution.

Antikinetoplastid SAR study in 3-nitroimidazopyridine series: Identification of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties.

To study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E° = -0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program.

8-Alkynyl-3-nitroimidazopyridines display potent antitrypanosomal activity against both T. b. brucei and cruzi.

An antikinetoplastid pharmacomodulation study was done at position 8 of a previously identified pharmacophore in 3-nitroimidazo[1,2-a]pyridine series. Twenty original derivatives bearing an alkynyl moiety were synthesized via a Sonogashira cross-coupling reaction and tested in vitro, highlighting 3 potent (40 nM ≤ EC50 blood stream form≤ 70 nM) and selective (500 ≤ SI ≤ 1800) anti-T. brucei brucei molecules (19, 21 and 22), in comparison with four reference drugs. Among these hit molecules, compound 19 also showed the same level of activity against T. cruzi (EC50 amastigotes = 1.2 µM) as benznidazole and fexinidazole. An in vitro comet assay showed that nitroaromatic derivative 19 was not genotoxic. It displayed a low redox potential value (-0.68 V/NHE) and was shown to be bioactivated by type 1 nitroreductases both in Leishmania and Trypanosoma. The SAR study indicated that an alcohol function improved aqueous solubility while maintaining good activity and low cytotoxicity when the hydroxyl group was at position beta of the alkyne triple bond. Hit-compound 19 was also evaluated regarding in vitro pharmacokinetic data: 19 is BBB permeable (PAMPA assay), has a 16 min microsomal half-life and a high albumin binding (98.5%). Moreover, compound 19 was orally absorbed and was well tolerated in mouse after both single and repeated administrations at 100 mg/kg. Its mouse plasma half-life (10 h) is also quite encouraging, paving the way toward further efficacy evaluations in parasitized mouse models, looking for a novel antitrypanosomal lead compound.

Antipsychotic lurasidone: Behavioural and pharmacokinetic data in C57BL/6 mice.

Lurasidone is an atypical antipsychotic that has been shown to be effective in reversing schizophrenia-related cognitive impairment. The development of new preclinical models of schizophrenia is a key for improving treatments of cognitive symptoms. This study investigated the effects of chronic lurasidone treatment in C57BL/6 male mice via intraperitoneal injection (1 mg/kg daily at 5 p.m. for 5 weeks). A large battery of behavioural tests was performed (between 9 a.m. and 5 p.m.), which is currently used to assess face validity in animal models of psychiatric diseases. Overall, lurasidone did not interfere with behavioural performances, which characterises very good tolerance to such a high dose. Moreover, pharmacokinetic parameters after i.p. and oral administration were measured. Mean transit time (MTT) values were 1.91 h (1 mg/kg acute i.p.) and 1.74 h (8.3 mg/kg acute oral), respectively, and relative bioavailability comparing these two routes of administration was of 19.8%. This last result gives important data to adapt oral chronic administration of lurasidone with a more ethical perspective in comparison with chronic i.p. injections. This study brings tools to improve pharmacological validity of preclinical models of psychiatric diseases, and to adapt dosage of antipsychotics according to the route used.

New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi.

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.

Pharmacotechnical Development of a Nasal Drug Delivery Composite Nanosystem Intended for Alzheimer's Disease Treatment.

Direct nose-to-brain delivery has been raised as a non-invasive powerful strategy to deliver drugs to the brain bypassing the blood-brain barrier (BBB). This study aimed at preparing and characterizing an innovative composite formulation, associating the liposome and hydrogel approaches, suitable for intranasal administration. Thermosensitive gel formulations were obtained based on a mixture of two hydrophilic polymers (Poloxamer 407, P407 and Poloxamer 188, P188) for a controlled delivery through nasal route via liposomes of an active pharmaceutical ingredient (API) of potential interest for Alzheimer's disease. The osmolarity and the gelation temperature (T° sol-gel) of formulations, defined in a ternary diagram, were investigated by rheometry and visual determination. Regarding the issue of assays, a mixture composed of P407/P188 (15/1%, w/w) was selected for intranasal administration in terms of T° sol-gel and for the compatibility with the olfactory mucosal (280 ± 20 mOsmol, pH 6). Liposomes of API were prepared by the thin film hydration method. Mucoadhesion studies were performed by using mucin disc, and they showed the good natural mucoadhesive characteristics of in situ gel formulations, which increased when liposomes were added. The study demonstrated successful pharmacotechnical development of a promising API-loaded liposomes in a thermosensitive hydrogel intended for nasal Alzheimer's disease treatment.

Synthesis of Pyridoclax Analogues: Insight into Their Druggability by Investigating Their Physicochemical Properties and Interactions with Membranes.

Pyridoclax is considered a promising anticancer drug, acting as a protein-protein interaction disruptor, with potential applications in the treatment of ovarian, lung, and mesothelioma cancers. Eighteen sensibly selected structural analogues of Pyridoclax were synthesized, and their physicochemical properties were systematically assessed and analyzed. Moreover, considering that drug-membrane interactions play an essential role in understanding the mode of action of a given drug and its eventual toxic effects, membrane models were used to investigate such interactions in bulk (liposomes) and at the air-water interface. The measured experimental data on all original oligopyridines allowed the assessment of relative differences in terms of physicochemical properties, which could be determinant for their druggability, and hence for drug development.

Novel multi target-directed ligands targeting 5-HT4 receptors with in cellulo antioxidant properties as promising leads in Alzheimer's disease.

Facing the complexity of Alzheimer's disease (AD), it is now currently admitted that a therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same time Aß protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells. Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical aminochlorobenzophenone present in numerous 5-HT4R agonists, and diverse antioxidant chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards 5-HT4R and an antioxidant activity in vitro and in cellulo.

Inhibiting Acetylcholinesterase to Activate Pleiotropic Prodrugs with Therapeutic Interest in Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which is still poorly understood. The drugs currently used against AD, mainly acetylcholinesterase inhibitors (AChEI), are considered clinically insufficient and are responsible for deleterious side effects. AChE is, however, currently receiving renewed interest through the discovery of a chaperone role played in the pathogenesis of AD. But AChE could also serve as an activating protein for pleiotropic prodrugs. Indeed, inhibiting central AChE with brain-penetrating designed carbamates which are able to covalently bind to the enzyme and to concomitantly liberate active metabolites in the brain could constitute a clinically more efficient approach which, additionally, is less likely to cause peripheral side effects. We aim in this article to pave the road of this new avenue with an in vitro and in vivo study of pleiotropic prodrugs targeting both the 5-HT4 receptor and AChE, in order to display a neuroprotective activity associated with a sustained restoration of the cholinergic neurotransmission and without the usual peripheral side effects associated with classic AChEI. This plural activity could bring to AD patients effective, relatively safe, symptomatic and disease-modifying therapeutic benefits.

Microplate assay for lipophilicity determination using intrinsic fluorescence of drugs: Application to a promising anticancer lead, pyridoclax.

Lipophilicity must be necessarily determined in drug discovery since this physicochemical property will directly influence the pharmacokinetics of a drug as its pharmacodynamics profile. Pyridoclax is an original lead, recently identified as very promising in treatment of chemoresistant cancers. The partition coefficient (Kp) of this anticancer drug was determined by microplate assays, well adapted in drug discovery, since being rapid, and requiring only poor drug amounts. The analytical approach was performed either by UV derivative spectrophotometry after validation thanks to a set of basic, neutral and acid reference substances, or originally, by raw fluorescence spectrophotometry by taking advantage of the pyridoclax intrinsic fluorescence. Large unilamellar vesicles (LUVs) were formulated from soybean-, egg-, or dipalmitoyl-phosphatidylcholine, characterized in terms of granulometric properties, ζ potential (determined by DLS), and of phospholipid content (quantified by 1H NMR, also in presence of cholesterol). Whatever the detection method used, log Kp of pyridoclax were in the same magnitude order, and pyridoclax appeared as a lipophilic compound. It was also established that interactions between this lead and biomimetic membranes were influenced by the relative fluidity of the membranes, as confirmed by results of a liposome leakage assay.