ChemoDOTS: a web server to design chemistry-driven focused libraries.

In drug discovery, the successful optimization of an initial hit compound into a lead molecule requires multiple cycles of chemical modification. Consequently, there is a need to efficiently generate synthesizable chemical libraries to navigate the chemical space surrounding the primary hit. To address this need, we introduce ChemoDOTS, an easy-to-use web server for hit-to-lead chemical optimization freely available at https://chemodots.marseille.inserm.fr/. With this tool, users enter an activated form of the initial hit molecule then choose from automatically detected reactive functions. The server proposes compatible chemical transformations via an ensemble of encoded chemical reactions widely used in the pharmaceutical industry during hit-to-lead optimization. After selection of the desired reactions, all compatible chemical building blocks are automatically coupled to the initial hit to generate a raw chemical library. Post-processing filters can be applied to extract a subset of compounds with specific physicochemical properties. Finally, explicit stereoisomers and tautomers are computed, and a 3D conformer is generated for each molecule. The resulting virtual library is compatible with most docking software for virtual screening campaigns. ChemoDOTS rapidly generates synthetically feasible, hit-focused, large, diverse chemical libraries with finely-tuned physicochemical properties via a user-friendly interface providing a powerful resource for researchers engaged in hit-to-lead optimization.

Cinacalcet Reverses Short QT Interval in Familial Hypocalciuric Hypercalcemia Type 1.

CONTEXT: Familial hypocalciuric hypercalcemia type 1 (FHH-1) defines an autosomal dominant disease, related to mutations in the CASR gene, with mild hypercalcemia in most cases. Cases of FHH-1 with a short QT interval have not been reported to date. OBJECTIVE: Three family members presented with FHH-1 and short QT interval (<360 ms), a condition that could lead to cardiac arrhythmias, and the effects of cinacalcet, an allosteric modulator of the CaSR, in rectifying the abnormal sensitivity of the mutant CaSR and in correcting the short QT interval were determined. METHODS: CASR mutational analysis was performed by next-generation sequencing and functional consequences of the identified CaSR variant (p.Ile555Thr), and effects of cinacalcet were assessed in HEK293 cells expressing wild-type and variant CaSRs. A cinacalcet test consisting of administration of 30 mg cinacalcet (8 Am) followed by hourly measurement of serum calcium, phosphate, and parathyroid hormone during 8 hours and an electrocardiogram was performed. RESULTS: The CaSR variant (p.Ile555Thr) was confirmed in all 3 FHH-1 patients and was shown to be associated with a loss of function that was ameliorated by cinacalcet. Cinacalcet decreased parathyroid hormone by >50% within two hours, and decreases in serum calcium and increases in serum phosphate occurred within 8 hours, with rectification of the QT interval, which remained normal after 3 months of cinacalcet treatment. CONCLUSION: Our results indicate that FHH-1 patients should be assessed for a short QT interval and a cinacalcet test used to select patients who are likely to benefit from this treatment.

Second sound resonators and tweezers as vorticity or velocity probes: Fabrication, model, and method.

An analytical model of open-cavity second sound resonators is presented and validated against simulations and experiments in superfluid helium using a new resonator design that achieves unprecedented resolution. The model incorporates diffraction, geometrical misalignments, and flow through the cavity and is validated using cavities operated up to their 20th resonance in superfluid helium. An important finding is that resonators can be optimized to selectively sense either the quantum vortex density carried by the throughflow-as typically done in the literature-or the mean velocity of the throughflow. We propose two velocity probing methods: one that takes advantage of misalignments between the tweezers' plates and other that drives the resonator non-linearly, beyond a threshold that results in the self-sustainment of a vortex tangle within the cavity. A new mathematical treatment of the resonant signal is proposed to adequately filter out parasitic signals, such as temperature and pressure drift, and accurately separate the quantum vorticity signal. This elliptic method consists in a geometrical projection of the resonance in the inverse complex plane. Its effectiveness is demonstrated over a wide range of operating conditions. The resonator model and elliptic method are being utilized to characterize a new design of resonators with high resolution, thanks to miniaturization and design optimization. These second-sound tweezers are capable of providing time-space resolved information similar to classical local probes in turbulence, down to sub-millimeter and sub-millisecond scales. The principle, design, and microfabrication of second sound tweezers are being presented, along with their potential for exploring quantum turbulence.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia.

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target. Herein, we describe the creation of a "best-in-class" sub-micromolar small molecule inhibitor for the second PDZ domain of syntenin validated in cancer cell assays. Key to the success of our DOTS approach was the integration of protein conformational sampling during hit identification stage and the synthetic feasibility ranking of the designed compounds throughout the optimization process. This approach can be broadly applied to other protein targets with known 3D structures to rapidly identify and optimize compounds as chemical probes and therapeutic candidates.

Discovery of Small-Molecule Inhibitors of the PTK7/ß-Catenin Interaction Targeting the Wnt Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC), the second cause of death due to cancer worldwide, is a major public health issue. The discovery of new therapeutic targets is thus essential. Pseudokinase PTK7 intervenes in the regulation of the Wnt/ß-catenin pathway signaling, in part, through a kinase domain-dependent interaction with the ß-catenin protein. PTK7 is overexpressed in CRC, an event associated with metastatic development and reduced survival of nonmetastatic patients. In addition, numerous alterations have been identified in CRC inducing constitutive activation of the Wnt/ß-catenin pathway signaling through ß-catenin accumulation. Thus, targeting the PTK7/ß-catenin interaction could be of interest for future drug development. We have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and ß-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small-molecule inhibitors targeting the Wnt pathway signaling and inducing antiproliferative and antitumor effects in vitro in CRC cells harboring ß-catenin or adenomatous polyposis coli (APC) mutations. Thus, inhibition of the PTK7/ß-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on the Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

CRCM5484: A BET-BDII Selective Compound with Differential Anti-leukemic Drug Modulation.

Differentially screening the Fr-PPIChem chemical library on the bromodomain and extra-terminal (BET) BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII-selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the bromodomain (BD) cavity and its potential as a starting scaffold for the development of potent and selective bromodomain inhibitors.

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

Rational Design of PDZ Domain Inhibitors: Discovery of Small Organic Compounds Targeting PDZ Domains.

PDZ domains, which belong to protein-protein interaction networks, are critical for regulating important biological processes such as scaffolding, trafficking, and signaling cascades. Interfering with PDZ-mediated interactions could affect these numerous biological processes. Thus, PDZ domains have emerged as promising targets to decipher biological phenomena and potentially treat cancer and neurological diseases. In this minireview, we focus on the discovery and design of small molecule inhibitors to modulate PDZ domains. These compounds interfere with endogenous protein partners from the PDZ domain by binding at the protein-protein interface. While peptides or peptidomimetic ligands were described to modulate PDZ domains, the focus of this review is on small organic compounds.

A gain-of-function RAC2 mutation is associated with bone-marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency.

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. They are defined by the absence of autologous T cells and the presence of an intrinsic or extrinsic defect in the B-cell compartment. In three newborns presenting with frequent infections and profound leukopenia, we identified a private, heterozygous mutation in the RAC2 gene (p.G12R). This mutation was de novo in the index case, who had been cured by hematopoietic stem cell transplantation but had transmitted the mutation to her sick daughter. Biochemical assays showed that the mutation was associated with a gain of function. The results of in vitro differentiation assays showed that RAC2 is essential for the survival and differentiation of hematopoietic stem/progenitor cells. Therefore, screening for RAC2 gain-of-function mutations should be considered in patients with a SCID phenotype and who lack a molecular diagnosis.

Emergency air evacuation of patients with acute respiratory failure due to SARS-CoV-2 from Mayotte to Reunion Island.

ABSTRACT: In February 2021, an explosion of cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia overwhelmed the only hospital in Mayotte. To report a case series of patients with acute respiratory failure (ARF) due to SARS-CoV-2 who were evacuated by air from Mayotte to Reunion Island.This retrospective observational study evaluated all consecutive patients with ARF due to SARS-CoV-2 who were evacuated by air from Mayotte Hospital to the intensive care unit (ICU) of Félix Guyon University Hospital in Reunion Island between February 2, and March 5, 2021.A total of 43 patients with SARS-CoV-2 pneumonia were evacuated by air, for a total flight time of 2 hours and a total travel time of 6 hours. Of these, 38 patients (88.4%) with a median age of 55 (46-65) years presented with ARF and were hospitalized in our ICU. Fifteen patients were screened for the SARS-CoV-2 501Y.V2 variant, all of whom tested positive. Thirteen patients (34.2%) developed an episode of severe hypoxemia during air transport, and the median paO2/FiO2 ratio was lower on ICU admission (140 [102-192] mmHg) than on departure (165 [150-200], P = .022). Factors associated with severe hypoxemia during air transport was lack of treatment with curare (P = .012) and lack of invasive mechanical ventilation (P = .003). Nine patients (23.7%) received veno-venous extracorporeal membrane oxygenation support in our ICU. Seven deaths (18.4%) occurred in hospital.Emergency air evacuation of patients with ARF due to SARS-CoV-2 was associated with severe hypoxemia but remained feasible. In cases of ARF due to SARS-CoV-2 requiring emergency air evacuation, sedated patients receiving invasive mechanical ventilation and curare should be prioritized over nonintubated patients. It is noteworthy that patients with SARS-CoV-2 pneumonia related to the 501Y.V2 variant were very severe despite their young age.

Fr-PPIChem: An Academic Compound Library Dedicated to Protein-Protein Interactions.

Protein-protein interactions (PPIs) mediate nearly every cellular process and represent attractive targets for modulating disease states but are challenging to target with small molecules. Despite this, several PPI inhibitors (iPPIs) have entered clinical trials, and a growing number of PPIs have become validated drug targets. However, high-throughput screening efforts still endure low hit rates mainly because of the use of unsuitable screening libraries. Here, we describe the collective effort of a French consortium to build, select, and store in plates a unique chemical library dedicated to the inhibition of PPIs. Using two independent predictive models and two updated databases of experimentally confirmed PPI inhibitors developed by members of the consortium, we built models based on different training sets, molecular descriptors, and machine learning methods. Independent statistical models were used to select putative PPI inhibitors from large commercial compound collections showing great complementarity. Medicinal chemistry filters were applied to remove undesirable structures from this set (such as PAINS, frequent hitters, and toxic compounds) and to improve drug likeness. The remaining compounds were subjected to a clustering procedure to reduce the final size of the library while maintaining its chemical diversity. In practice, the library showed a 46-fold activity rate enhancement when compared to a non-iPPI-enriched diversity library in high-throughput screening against the CD47-SIRPα PPI. The Fr-PPIChem library is plated in 384-well plates and will be distributed on demand to the scientific community as a powerful tool for discovering new chemical probes and early hits for the development of potential therapeutic drugs.

A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.

Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

CovaDOTS: In Silico Chemistry-Driven Tool to Design Covalent Inhibitors Using a Linking Strategy.

We recently reported an integrated fragment-based optimization strategy called DOTS (Diversity Oriented Target-focused Synthesis) that combines automated virtual screening (VS) with semirobotized organic synthesis coupled to in vitro evaluation. The molecular modeling part consists of hit-to-lead chemistry, based on the growing paradigm. Here, we have extended the applicability of the DOTS strategy by adding new functionalities, allowing a generic chemistry-driven linking approach with a particular emphasis on covalent drugs. Indeed, the covalent mode of action can be described as a specific case of linking, where suitable linkers are sought to fuse a bound organic compound with a nucleophilic protein side chain. The proof of concept is established using three retrospective study cases in which known noncovalent inhibitors have been converted to covalent inhibitors. Our method is able to automatically design reference covalent inhibitors (and/or analogs) from an initial activated substructure and predict their binding mode. More importantly, the reference compounds are ranked high among several hundred putative adducts, demonstrating the utility of the approach to design covalent inhibitors.

Optimization of a fragment linking hit toward Dengue and Zika virus NS5 methyltransferases inhibitors.

No antiviral drugs to treat or prevent life-threatening flavivirus infections such as those caused by mosquito-borne Dengue (DENV) and more recently Zika (ZIKV) viruses are yet available. We aim to develop, through a structure-based drug design approach, novel inhibitors targeting the NS5 AdoMet-dependent mRNA methyltransferase (MTase), a viral protein involved in the RNA capping process essential for flaviviruses replication. Herein, we describe the optimization of a hit (5) identified using fragment-based and structure-guided linking techniques, which binds to a proximal site of the AdoMet binding pocket. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 30 and 33 (DENV IC50 = 26 µM and 23 µM; ZIKV IC50 = 28 µM and 19  µM, respectively), two representatives of novel non-nucleoside inhibitors of flavivirus MTases.

Chemistry-driven Hit-to-lead Optimization Guided by Structure-based Approaches.

For several decades, hit identification for drug discovery has been facilitated by developments in both fragment-based and high-throughput screening technologies. However, a major bottleneck in drug discovery projects continues to be the optimization of primary hits from screening campaigns in order to derive lead compounds. Computational chemistry or molecular modeling can play an important role during this hit-to-lead (H2L) stage by both suggesting putative optimizations and decreasing the number of compounds to be experimentally synthesized and evaluated. However, it is also crucial to consider the feasibility of organically synthesizing these virtually designed compounds. Furthermore, the generated molecules should have reasonable physicochemical properties and be medicinally relevant. This review focuses on chemistry-driven and structure-based computational methods that can be used to tackle the difficult problem of H2L optimization, with emphasis being placed on the strategy developed in our laboratory.

Integrated Strategy for Lead Optimization Based on Fragment Growing: The Diversity-Oriented-Target-Focused-Synthesis Approach.

Over the past few decades, hit identification has been greatly facilitated by advances in high-throughput and fragment-based screenings. One major hurdle remaining in drug discovery is process automation of hit-to-lead (H2L) optimization. Here, we report a time- and cost-efficient integrated strategy for H2L optimization as well as a partially automated design of potent chemical probes consisting of a focused-chemical-library design and virtual screening coupled with robotic diversity-oriented de novo synthesis and automated in vitro evaluation. The virtual library is generated by combining an activated fragment, corresponding to the substructure binding to the target, with a collection of functionalized building blocks using in silico encoded chemical reactions carefully chosen from a list of one-step organic transformations relevant in medicinal chemistry. The proof of concept was demonstrated using the optimization of bromodomain inhibitors as a test case, leading to the validation of several compounds with improved affinity by several orders of magnitude.

18F-FDOPA PET/CT Imaging of MAX-Related Pheochromocytoma.

Context: MYC-associated factor X (MAX) has been recently described as a new susceptibility pheochromocytoma (PHEO) gene with a total of ~40 reported cases. At present, no study has specifically described the functional imaging phenotype of MAX-related PHEO. Objective, Patients, and Design: The objective of the present study was to present our experience with contrast-enhanced computed tomography (CT) and 18F-fluorodihydroxyphenylalanine (18F-FDOPA) positron emission tomography (PET)/CT in six consecutive patients (four at the initial diagnosis and two at the follow-up evaluation) with rare, but clinically important, MAX-related PHEOs. In five patients, 18F-FDOPA was also compared with other radiopharmaceutical agents. Results: The patients had five different mutations in the MAX gene that caused disruption of Max/Myc interaction and/or abolished interaction with DNA based on in silico analyses. All but one patient developed bilateral PHEOs during their lifetime. In all cases, 18F-FDOPA PET/CT accurately visualized PHEOs that were often multiple within the same gland or bilaterally and detected more adrenal and extra-adrenal lesions than did CT (per-lesion sensitivity, 90.9% vs 52.4% for CT/magnetic resonance imaging). The two PHEOs missed on 18F-FDOPA PET/CT were <1 cm, corresponding to nodular adrenomedullary hyperplasia. 68Ga-DOTA,Tyr3-octreotate PET/CT detected fewer lesions than did 18F-FDOPA PET/CT in one of three patients, and 18F-fluorodeoxyglucose PET/CT was only faintly positive in two of four patients with underestimation of extra-adrenal lesions in one patient. Conclusions: MAX-related PHEOs exhibit a marked 18F-FDOPA uptake, a finding that illustrates the common well-differentiated chromaffin pattern of PHEOs associated with activation of kinase signaling pathways. 18F-FDOPA PET/CT should be considered as the first-line functional imaging modality for diagnostic or follow-up evaluations for these patients.