Discovery of 2-(3-Benzamidopropanamido)thiazole-5-carboxylate Inhibitors of the Kinesin HSET (KIFC1) and the Development of Cellular Target Engagement Probes.

The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans-cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

Binding to an Unusual Inactive Kinase Conformation by Highly Selective Inhibitors of Inositol-Requiring Enzyme 1α Kinase-Endoribonuclease.

A series of imidazo[1,2- b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766).

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.

Kernel-Based, Partial Least Squares Quantitative Structure-Retention Relationship Model for UPLC Retention Time Prediction: A Useful Tool for Metabolite Identification.

We propose a new QSRR model based on a Kernel-based partial least-squares method for predicting UPLC retention times in reversed phase mode. The model was built using a combination of classical (physicochemical and topological) and nonclassical (fingerprints) molecular descriptors of 1383 compounds, encompassing different chemical classes and structures and their accurately measured retention time values. Following a random splitting of the data set into a training and a test set, we tested the ability of the model to predict the retention time of all the compounds. The best predicted/experimental R2 value was higher than 0.86, while the best Q2 value we observed was close to 0.84. A comparison of our model with traditional and simpler MLR and PLS regression models shows that KPLS better performs in term of correlation (R2), prediction (Q2), and support to MetID peak assignment. The KPLS model succeeded in two real-life MetID tasks by correctly predicting elution order of Phase I metabolites, including isomeric monohydroxylated compounds. We also show in this paper that the model's predictive power can be extended to different gradient profiles, by simple mathematical extrapolation using a known equation, thus offering very broad flexibility. Moreover, the current study includes a deep investigation of different types of chemical descriptors used to build the structure-retention relationship.

O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

Synthesis and structure-activity relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid amide hydrolase inhibitors.

The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3'-carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.

Computer-aided identification, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus.

Chikungunya virus (CHIKV) is an Arbovirus that is transmitted to humans primarily by the mosquito species Aedes aegypti. Infection with this pathogen is often associated with fever, rash and arthralgia. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. Albeit considered a tropical pathogen, adaptation of the virus to the mosquito species Aedes albopictus, which is also very common in temperate zones, has resulted in recent outbreaks in Europe and the US. In the present study, we report on the discovery of a novel series of compounds that inhibit CHIKV replication in the low µM range. In particular, we initially performed a virtual screening simulation of ∼5 million compounds on the CHIKV nsP2, the viral protease, after which we investigated and explored the Structure-Activity Relationships of the hit identified in silico. Overall, a series of 26 compounds, including the original hit, was evaluated in a virus-cell-based CPE reduction assay. The study of such selective inhibitors will contribute to a better understanding of the CHIKV replication cycle and may represents a first step towards the development of a clinical candidate drug for the treatment of this disease.

Peripheral FAAH inhibition causes profound antinociception and protects against indomethacin-induced gastric lesions.

Fatty-acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide and other bioactive lipid amides. In the present study, we conducted a comparative characterization of the effects of the newly identified brain-impermeant FAAH inhibitor, URB937 ([3-(3-carbamoylphenyl)-4-hydroxy-phenyl] N-cyclohexylcarbamate), in various rodent models of acute and persistent pain. When administered by the oral route in mice, URB937 was highly active (median effective dose, ED(50), to inhibit liver FAAH activity: 0.3mgkg(-1)) and had a bioavailability of 5.3%. The antinociceptive effects of oral URB937 were investigated in mouse models of acute inflammation (carrageenan), peripheral nerve injury (chronic sciatic nerve ligation) and arthritis (complete Freund's adjuvant). In all models, URB937 was as effective or more effective than standard analgesic and anti-inflammatory drugs (indomethacin, gabapentin, dexamethasone) and reversed pain-related responses (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) in a dose-dependent manner. ED(50) values ranged from 0.2 to 10mgkg(-1), depending on model and readout. Importantly, URB937 was significantly more effective than two global FAAH inhibitors, URB597 and PF-04457845, in the complete Freund's adjuvant model. The effects of a combination of URB937 with the non-steroidal anti-inflammatory agent, indomethacin, were examined in the carrageenan and chronic sciatic nerve ligation models. Isobolographic analyses showed that the two compounds interacted synergistically to attenuate pain-related behaviors. Furthermore, URB937 reduced the number and severity of gastric lesions produced by indomethacin, while exerting no ulcerogenic effect when administered alone. The results indicate that the peripheral FAAH inhibitor URB937 is more effective than globally active FAAH inhibitors at inhibiting inflammatory pain. Our findings further suggest that FAAH and cyclooxygenase inhibitors interact functionally in peripheral tissues, to either enhance or hinder each other's actions.

A novel α-isocyanoacetamide-based three-component reaction for the synthesis of dialkyl 2-acyl-5-aminofuran-3,4-dicarboxylates.

α-Isocyanoacetamides, acyl chlorides and dialkylacetylenedicarboxylates undergo a smooth multicomponent reaction to produce dialkyl 2-acyl-5-aminofuran-3,4-dicarboxylates in good yield. The scope and mechanism of this new multicomponent transformation are discussed.

A novel potent nicotinamide phosphoribosyltransferase inhibitor synthesized via click chemistry.

The inhibition of NAD synthesis or salvage pathways has been proposed as a novel target for antitumoral drugs. Two molecules with this mechanism of action are at present undergoing clinical trials. In searching for similar novel molecules, we exploited copper-catalyzed [3 + 2] cycloaddition between azides and alkynes (click chemistry) to synthesize 185 novel analogues. The most promising compound displays an IC(50) for cytotoxicity in vitro of 3.8 +/- 0.3 nM and an IC(50) for NAD depletion of 3.0 +/- 0.4 nM. Herein, we strengthen previous data suggesting that this class of compounds induces autophagic cell death. In addition to characterizing this compound and providing a rationale via molecular docking, we reinforce the excellent potential of click chemistry for rapidly generating structure-activity relationships and for drug screening.

SERCA-inhibiting activity of C-19 terpenolides from Thapsia garganica and their possible biogenesis.

An investigation of Thapsia garganica afforded a series of tetracyclic C-19 dilactones, whose production was dependent on the time and location of the collection. These unusual tetrahomosesquiterpenoids are presumably biosynthesized via a carbon dioxide-triggered electrophilic polyolefin cyclization. Despite the structural differences with thapsigargin, these compounds showed SERCA-inhibiting properties.