Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K.

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation ( kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

Drug Discovery Maps, a Machine Learning Model That Visualizes and Predicts Kinome-Inhibitor Interaction Landscapes.

The interpretation of high-dimensional structure-activity data sets in drug discovery to predict ligand-protein interaction landscapes is a challenging task. Here we present Drug Discovery Maps (DDM), a machine learning model that maps the activity profile of compounds across an entire protein family, as illustrated here for the kinase family. DDM is based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm to generate a visualization of molecular and biological similarity. DDM maps chemical and target space and predicts the activities of novel kinase inhibitors across the kinome. The model was validated using independent data sets and in a prospective experimental setting, where DDM predicted new inhibitors for FMS-like tyrosine kinase 3 (FLT3), a therapeutic target for the treatment of acute myeloid leukemia. Compounds were resynthesized, yielding highly potent, cellularly active FLT3 inhibitors. Biochemical assays confirmed most of the predicted off-targets. DDM is further unique in that it is completely open-source and available as a ready-to-use executable to facilitate broad and easy adoption.

Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

A natural-product switch for a dynamic protein interface.

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.

Structure-Activity Relationship Studies of Aryl Sulfoxides as Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is the key enzyme for the hydrolysis of endocannabinoid 2-arachidonoylglycerol (2-AG). The central role of MAGL in the metabolism of 2-AG makes it an attractive therapeutic target for a variety of disorders, including inflammation-induced tissue injury, pain, multiple sclerosis, and cancer. Previously, we reported LEI-515, an aryl sulfoxide, as a peripherally restricted, covalent reversible MAGL inhibitor that reduced neuropathic pain and inflammation in preclinical models. Here, we describe the structure-activity relationship (SAR) of aryl sulfoxides as MAGL inhibitors that led to the identification of LEI-515. Optimization of the potency of high-throughput screening (HTS) hit 1 yielded compound ±43. However, ±43 was not metabolically stable due to its ester moiety. Replacing the ester group with α-CF2 ketone led to the identification of compound ±73 (LEI-515) as a metabolically stable MAGL inhibitor with subnanomolar potency. LEI-515 is a promising compound to harness the therapeutic potential of MAGL inhibition.

Discovery of isoquinoline sulfonamides as allosteric gyrase inhibitors with activity against fluoroquinolone-resistant bacteria.

Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase-LEI-800-DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones.

Epi-Cyclophellitol Cyclosulfate, a Mechanism-Based Endoplasmic Reticulum α-Glucosidase II Inhibitor, Blocks Replication of SARS-CoV-2 and Other Coronaviruses.

The combined inhibition of endoplasmic reticulum (ER) α-glucosidases I and II has been shown to inhibit replication of a broad range of viruses that rely on ER protein quality control. We found, by screening a panel of deoxynojirimycin and cyclitol glycomimetics, that the mechanism-based ER α-glucosidase II inhibitor, 1,6-epi-cyclophellitol cyclosulfate, potently blocks SARS-CoV-2 replication in lung epithelial cells, halting intracellular generation of mature spike protein, reducing production of infectious progeny, and leading to reduced syncytium formation. Through activity-based protein profiling, we confirmed ER α-glucosidase II inhibition in primary airway epithelial cells, grown at the air-liquid interface. 1,6-epi-Cyclophellitol cyclosulfate inhibits early pandemic and more recent SARS-CoV-2 variants, as well as SARS-CoV and MERS-CoV. The reported antiviral activity is comparable to the best-in-class described glucosidase inhibitors, all competitive inhibitors also targeting ER α-glucosidase I and other glycoprocessing enzymes not involved in ER protein quality control. We propose selective blocking ER-resident α-glucosidase II in a covalent and irreversible manner as a new strategy in the search for effective antiviral agents targeting SARS-CoV-2 and other viruses that rely on ER protein quality control.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Structure-Activity Relationship Studies of Pyrimidine-4-Carboxamides as Inhibitors of N-Acylphosphatidylethanolamine Phospholipase D.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [Mock Nat Chem. Biol., 2020, 16, 667-675]. Here, we describe the structure-activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

Synthetic heparin derivatives as new anticoagulant drugs.

The journey towards a detailed mechanistic understanding of the anticoagulant action of heparin has resulted in synthetic mimetics with improved pharmacodynamic profiles. Inspired by the ternary complex formation of heparin with antithrombin III and thrombin, the active pentasaccharide fondaparinux has been succeeded by several clinical candidates, such as SR123781, that have tailor-made factor Xa and thrombin inhibitory activities combined with less aspecific binding (e.g. binding to platelet factor 4 involved in thrombocytopenia). Novel compounds with both antithrombin III-mediated inhibition of factor Xa and direct thrombin inhibition are emerging. Org42675 is one such compound, balancing dual inhibition of factor Xa and thrombin in one anticoagulant drug, with excellent pharmacokinetic properties and strong inhibitory activity toward clot-bound thrombin.

Targeting RNA: new opportunities to address drugless targets.

Historically, pharmaceutical industries have focussed on the discovery of compounds that target the protein products of genes. The intermediary product between gene and protein, consisting of RNA, has remained largely unexplored. Several drugs targeting the rRNA of bacteria have been, however, in clinical use for over half a century. One of these drug classes, the aminoglycoside antibiotics, also targets human rRNA, and have been developed as therapeutics for genetic disorders. Targeting at the RNA level is an economical approach to address non-drugable proteins and targets that have failed to give leads by hits in HTS, as it can build on biological knowledge gathered over years. RNA also offers entirely new opportunities for drug development, such as targeting of non-coding RNA sequences.

A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next?

Heparin is a sulfated glycosaminoglycan isolated from animal organs that has been used clinically as an antithrombotic agent since the 1940s. In the early 1980s it was discovered that a unique pentasaccharide domain in some heparin chains activates antithrombin III (AT-III), a serine protease inhibitor that blocks thrombin and factor Xa in the coagulation cascade. Sanofi-Synthélabo and Organon developed a synthetic analogue of this pentasaccharide. The resulting antithrombotic drug arixtra, which went on the market in the USA and Europe in 2002, shows superior antithrombotic activity and brings about AT-III-mediated activity against factor Xa exclusively. Structure-based design has subsequently led to analogues with longer-lasting activity, such as idraparinux, as well as novel conjugates and long oligosaccharides with specific anti-Xa and antithrombin activities. The new drug candidates are more selective in their mode of action than heparin and less likely to induce thrombocytopenia.

Unique overlap in the prerequisites for thrombin inhibition and oral bioavailability resulting in potent oral antithrombotics.

Despite intense research over the last 10 years, aided by the availability of X-ray structures of enzyme-inhibitor complexes, only very few truly orally active thrombin inhibitors have been found. We conducted a comprehensive study starting with peptide transition state analogues (TSA). Both hydrophobic nonpeptide analogues as well as hydrophilic peptidic analogues were synthesized. The bioavailability in rats and dogs could be drastically altered depending on the overall charge distribution in the molecule. Compound 27, a tripeptide TSA inhibitor of thrombin, showed an oral bioavailability of 32% in rats and 71% in dogs, elimination half-lives being 58 and 108 min, respectively. The thrombin inhibition constant of compound 27 was 1.1 nM, and in an in vivo arterial flow model, the ED(50) was 5.4 nmol/kg.min, comparable to known non-TSA inhibitors. A molecular design was found that combines antithrombotic efficiency with oral bioavailability at low dosages.

The synthesis of well-defined heparin and heparan sulfate fragments.

Heparin and heparan sulfate are key players in a plethora of physiological processes. Organic synthesis is the method of choice for the production of these oligosaccharides and their derivatives and analogues. The highly complex structure of these polysaccharides presents a formidable synthetic challenge and the incorporation of the full array of variations in oligosaccharides of significant length is a daunting task. This review records the development of strategies to access these exciting biomolecules.:

First Synthetic Carbohydrates with the Full Anticoagulant Properties of Heparin.

A single disaccharide building block is required to obtain synthetic carbohydrates that reproduce the anticoagulant activity of heparin and inhibit thrombin (n>6) and/or factor Xa (n≥2; see reaction scheme). Thus, there is evidence that heparin fragments with at least 15 saccharide units are required for thrombin inhibition. Lev=levulinoyl.

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index.

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

From heparin to EP217609: the long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile.

The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.