Chemical, Biochemical, Cellular, and Physiological Characterization of Leucettinib-21, a Down Syndrome and Alzheimer's Disease Drug Candidate.

Leucettinibs are substituted 2-aminoimidazolin-4-ones (inspired by the marine sponge natural product Leucettamine B) developed as pharmacological inhibitors of DYRK1A (dual-specificity, tyrosine phosphorylation-regulated kinase 1A), a therapeutic target for indications such as Down syndrome and Alzheimer's disease. Leucettinib-21 was selected as a drug candidate following extensive structure/activity studies and multiparametric evaluations. We here report its physicochemical properties (X-ray powder diffraction, differential scanning calorimetry, stability, solubility, crystal structure) and drug-like profile. Leucettinib-21's selectivity (analyzed by radiometric, fluorescence, interaction, thermal shift, residence time assays) reveals DYRK1A as the first target but also some "off-targets" which may contribute to the drug's biological effects. Leucettinib-21 was cocrystallized with CLK1 and modeled in the DYRK1A structure. Leucettinib-21 inhibits DYRK1A in cells (demonstrated by direct catalytic activity and phosphorylation levels of Thr286-cyclin D1 or Thr212-Tau). Leucettinib-21 corrects memory disorders in the Down syndrome mouse model Ts65Dn and is now entering safety/tolerance phase 1 clinical trials.

Affinity selection mass spectrometry speeding drug discovery.

Affinity selection mass spectrometry (AS-MS) has gained momentum in drug discovery. This review summarizes how this technology has slowly risen as a new paradigm in hit identification and its potential synergy with DNA encoded library technology. It presents an overview of the recent results on challenging targets and perspectives on new areas of research, such as RNA targeting with small molecules. The versatility of the approach is illustrated and strategic drivers discussed in terms of the experience of a small-medium CRO and a big pharma organization.

Leucettinibs, a Class of DYRK/CLK Kinase Inhibitors Inspired by the Marine Sponge Natural Product Leucettamine B.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) recently attracted attention due to their central involvement in various pathologies. We here describe a family of DYRK/CLK inhibitors derived from Leucettines and the marine natural product Leucettamine B. Forty-five N2-functionalized 2-aminoimidazolin-4-ones bearing a fused [6 + 5]-heteroarylmethylene were synthesized. Benzothiazol-6-ylmethylene was selected as the most potent residue among 15 different heteroarylmethylenes. 186 N2-substituted 2-aminoimidazolin-4-ones bearing a benzothiazol-6-ylmethylene, collectively named Leucettinibs, were synthesized and extensively characterized. Subnanomolar IC50 (0.5-20 nM on DYRK1A) inhibitors were identified and one Leucettinib was modeled in DYRK1A and co-crystallized with CLK1 and the weaker inhibited off-target CSNK2A1. Kinase-inactive isomers of Leucettinibs (>3-10 µM on DYRK1A), named iso-Leucettinibs, were synthesized and characterized as suitable negative control compounds for functional experiments. Leucettinibs, but not iso-Leucettinibs, inhibit the phosphorylation of DYRK1A substrates in cells. Leucettinibs provide new research tools and potential leads for further optimization toward therapeutic drug candidates.

Therapeutic potential of compounds targeting SARS-CoV-2 helicase.

The economical and societal impact of COVID-19 has made the development of vaccines and drugs to combat SARS-CoV-2 infection a priority. While the SARS-CoV-2 spike protein has been widely explored as a drug target, the SARS-CoV-2 helicase (nsp13) does not have any approved medication. The helicase shares 99.8% similarity with its SARS-CoV-1 homolog and was shown to be essential for viral replication. This review summarizes and builds on existing research on inhibitors of SARS-CoV-1 and SARS-CoV-2 helicases. Our analysis on the toxicity and specificity of these compounds, set the road going forward for the repurposing of existing drugs and the development of new SARS-CoV-2 helicase inhibitors.

Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Exploring new targets and chemical space with affinity selection-mass spectrometry.

Affinity selection-mass spectrometry (AS-MS) is a high-throughput screening (HTS) technique for drug discovery that enables rapid screening of large collections of compounds to identify ligands for a specific biomolecular target. AS-MS is a binding assay that is insensitive to the functional effects a ligand might have, which is important because it lets us identify novel ligands irrespective of their binding site. This approach is gaining popularity, notably due to its role in the emergence of useful agents for targeted protein degradation. This Perspective highlights the use of AS-MS techniques to explore broad chemical space and identify small-molecule ligands for biological targets that have proven challenging to address with other screening paradigms. We present chemical structures of reported AS-MS hits to illustrate the potential of this screening approach to deliver high-quality hits for further optimization. AS-MS has, thus, evolved from being an infrequent alternative to traditional HTS or DNA-encoded library strategies to now firmly establishing itself as a HTS approach for drug discovery.

Novel 2,7-Diazaspiro[4,4]nonane Derivatives to Inhibit Mouse and Human Osteoclast Activities and Prevent Bone Loss in Ovariectomized Mice without Affecting Bone Formation.

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N-arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Interglacial instability of North Atlantic Deep Water ventilation.

Disrupting North Atlantic Deep Water (NADW) ventilation is a key concern in climate projections. We use (sub)centennially resolved bottom water δ13C records that span the interglacials of the last 0.5 million years to assess the frequency of and the climatic backgrounds capable of triggering large NADW reductions. Episodes of reduced NADW in the deep Atlantic, similar in magnitude to glacial events, have been relatively common and occasionally long-lasting features of interglacials. NADW reductions were triggered across the range of recent interglacial climate backgrounds, which demonstrates that catastrophic freshwater outburst floods were not a prerequisite for large perturbations. Our results argue that large NADW disruptions are more easily achieved than previously appreciated and that they occurred in past climate conditions similar to those we may soon face.

Discovery of novel Cyclophilin D inhibitors starting from three dimensional fragments with millimolar potencies.

Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.

A two-million-year-long hydroclimatic context for hominin evolution in southeastern Africa.

The past two million years of eastern African climate variability is currently poorly constrained, despite interest in understanding its assumed role in early human evolution1-4. Rare palaeoclimate records from northeastern Africa suggest progressively drier conditions2,5 or a stable hydroclimate6. By contrast, records from Lake Malawi in tropical southeastern Africa reveal a trend of a progressively wetter climate over the past 1.3 million years7,8. The climatic forcings that controlled these past hydrological changes are also a matter of debate. Some studies suggest a dominant local insolation forcing on hydrological changes9-11, whereas others infer a potential influence of sea surface temperature changes in the Indian Ocean8,12,13. Here we show that the hydroclimate in southeastern Africa (20-25° S) is controlled by interplay between low-latitude insolation forcing (precession and eccentricity) and changes in ice volume at high latitudes. Our results are based on a multiple-proxy reconstruction of hydrological changes in the Limpopo River catchment, combined with a reconstruction of sea surface temperature in the southwestern Indian Ocean for the past 2.14 million years. We find a long-term aridification in the Limpopo catchment between around 1 and 0.6 million years ago, opposite to the hydroclimatic evolution suggested by records from Lake Malawi. Our results, together with evidence of wetting at Lake Malawi, imply that the rainbelt contracted toward the Equator in response to increased ice volume at high latitudes. By reducing the extent of woodland or wetlands in terrestrial ecosystems, the observed changes in the hydroclimate of southeastern Africa-both in terms of its long-term state and marked precessional variability-could have had a role in the evolution of early hominins, particularly in the extinction of Paranthropus robustus.

Translation of innovative chemistry into screening libraries: an exemplar partnership from the European Lead Factory.

The identification of high-quality starting points for drug discovery is an enduring challenge in medicinal chemistry. Yet, the chemical space explored in discovery programmes tends be limited by the narrow toolkit of robust methods that are exploited in discovery workflows. The European Lead Factory (ELF) was established in 2013 to boost early-stage drug discovery within Europe. In this Feature, we describe an exemplar partnership that has led to the addition of 21119 distinctive screening compounds to the ELF Joint European Compound Library. The partnership could serve as a blueprint for the translation of innovative academic chemistry into discovery programmes.

Rational Drug Design of Topically Administered Caspase 1 Inhibitors for the Treatment of Inflammatory Acne.

The use of an interleukin ß antibody is currently being investigated in the clinic for the treatment of acne, a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1ß into active IL-1ß, caspase-1, could be an alternative small molecule approach. This report describes the discovery of uracil 20, a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration, topical administration challenges such as phototoxicity, organic and aqueous solubility, chemical stability in solution, and skin metabolic stability are discussed and successfully resolved.

New Caspase-1 inhibitor by scaffold hopping into bio-inspired 3D-fragment space.

Virtual fragmentation of a library of 12,000 compounds inspired by natural products led to a dataset of 153,000 fragments that was used as a source to identify effective P2-P3 scaffold replacement solutions for peptidic Caspase-1 inhibitors. Our strategy led to the identification of an original 2-azabicyclo-octane scaffold (2-ABO) that was further elaborated into the potent Caspase-1 inhibitor CD10847 (IC50 = 17 nM). The crystal structure of Caspase-1 in complex with CD10847 was obtained, and its binding mode was shown to be similar to the one predicted by docking and in good agreement with other known inhibitors.

Rapid termination of the African Humid Period triggered by northern high-latitude cooling.

The rapidity and synchrony of the African Humid Period (AHP) termination at around 5.5 ka are debated, and it is unclear what caused a rapid hydroclimate response. Here we analysed the hydrogen isotopic composition of sedimentary leaf-waxes (δDwax) from the Gulf of Guinea, a proxy for regional precipitation in Cameroon and the central Sahel-Sahara. Our record indicates high precipitation during the AHP followed by a rapid decrease at 5.8-4.8 ka. The similarity with a δDwax record from northern East Africa suggests a large-scale atmospheric mechanism. We show that northern high- and mid-latitude cooling weakened the Tropical Easterly Jet and, through feedbacks, strengthened the African Easterly Jet. The associated decrease in precipitation triggered the AHP termination and combined with biogeophysical feedbacks to result in aridification. Our findings suggest that extratropical temperature changes, albeit smaller than during the glacial and deglacial, were important in triggering rapid African aridification during the Holocene.

Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action.

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Design, synthesis and decoration of molecular scaffolds for exploitation in the production of alkaloid-like libraries.

The design, synthesis and decoration of six small molecule libraries is described. Each library was inspired by structures embedded in the framework of specific alkaloid natural products. The development of optimised syntheses of the required molecular scaffolds is described, in which reactions including Pd-catalysed aminoarylation and diplolar cycloadditions have been exploited as key steps. The synthesis of selected exemplar screening compounds is also described. In five cases, libraries were subsequently nominated for production on the basis of the scope and limitations of the validation work, as well as predicted molecular properties. In total, the research has led to the successful synthesis of >2500 novel alkaloid-like compounds for addition to the screening collection (the Joint European Compound Library, JECL) of the European Lead Factory.

Orbital Asian summer monsoon dynamics revealed using an isotope-enabled global climate model.

The Asian summer monsoon dynamics at the orbital scale are a subject of considerable debate. The validity of Asian speleothem δ(18)O records as a proxy for summer monsoon intensity is questioned together with the ultimate forcing and timing of the monsoon. Here, using the results of a 150,000-year transient simulation including water isotopes, we demonstrate that Asian speleothem δ(18)O records are not a valid proxy for summer monsoon intensity only at the orbital timescale. Rather, our results show that these records reflect annual variations in hydrologic processes and circulation regime over a large part of the Indo-Asian region. Our results support the role of internal forcing, such as sea surface temperature in the equatorial Pacific, to modulate the timing of monsoon precipitation recorded in paleo-proxies inside the Asian region.

The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.