High‐Throughput Virtual Screening Using Quantum Mechanical Probes: Discovery of Selective Kinase Inhibitors

A procedure based on semi‐empirical quantum mechanical (QM) calculations of interaction energy is proposed for the rapid screening of compound poses generated by high‐throughput docking. Small molecules (consisting of 2–10 atoms and termed “probes”) are overlapped with polar groups in the binding site of the protein target. The interaction energy values between each compound pose and the probes, calculated by a semi‐empirical Hamiltonian, are used as filters. The QM probe method does not require fixed partial charges and takes into account polarization and charge‐transfer effects which are not captured by conventional force fields. The procedure is applied to screen ～100 million poses (of 2.7 million commercially available compounds) obtained by high‐throughput docking in the ATP binding site of the tyrosine kinase erythropoietin‐producing human hepatocellular carcinoma receptor B4 (EphB4). Three QM probes on the hinge region and one at the entrance pocket are employed to select for binding affinity, while a QM probe on the side chain of the so‐called gatekeeper residue (a hypervariable residue in the kinome) is used to enforce selectivity. The poses with favorable interactions with the five QM probes are filtered further for hydrophobic matching and low ligand strain. In this way, a single‐digit micromolar inhibitor of EphB4 with a relatively good selectivity profile is identified in a multimillion‐compound library upon experimental tests of only 23 molecules.     

On‐Chip Fragment‐Based Approach for Discovery of High‐Affinity Bivalent Inhibitors

Covalent bonds not required : We describe a novel approach in which the concepts of fragment‐based ligand discovery are combined with chemical array techniques to yield bivalent inhibitors. A pair of fragments is mixed and covalently attached to a glass slide by photolinking immobilization. The method does not require the compounds to have specific functional groups, and tedious steps for protein purification are avoided. Thus, the on‐chip fragment‐based approach is relatively simple and efficient for obtaining high‐affinity ligands.

     

Development of a Method for the High‐Throughput Quantification of Cellular Proteins

Hunting for huntingtin : We describe a screening assay based on the inducible expression of the mutant huntingtin protein in cells and on its highly sensitive homogenous determination. Rapid, reproducible, and robust protein determination was achieved through the use of two donor–acceptor‐labeled antibodies and time‐resolved FRET. The assay was developed and validated for ultra‐throughput screening of low‐molecular‐weight compounds modulating the expression of the mutant protein.

     

5‐Benzylidenethiazolidin‐4‐ones as Multitarget Inhibitors of Bacterial Mur Ligases

Mur ligases participate in the intracellular path of bacterial peptidoglycan biosynthesis and constitute attractive, although so far underexploited, targets for antibacterial drug discovery. A series of hydroxy‐substituted 5‐benzylidenethiazolidin‐4‐ones were synthesized and tested as inhibitors of Mur ligases. The most potent compound 5 a was active against MurD–F with IC₅₀ values between 2 and 6 μm, making it a promising multitarget inhibitor of Mur ligases. Antibacterial activity against different strains, inhibitory activity against protein kinases, mutagenicity and genotoxicity of 5 a were also investigated, and kinetic and NMR studies were conducted.     

Aryloxyethyl Thiocyanates Are Potent Growth Inhibitors of Trypanosoma cruzi and Toxoplasma gondii

As a part of our project aimed at searching for new safe chemotherapeutic agents against parasitic diseases, several compounds structurally related to the antiparasitic agent WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), which were modified at the terminal phenyl ring, were designed, synthesized, and evaluated as growth inhibitors against Trypanosoma cruzi, the etiological agent of Chagas disease, and Toxoplasma gondii, the parasite responsible of toxoplasmosis. Most of the synthetic analogues exhibited similar antiparasitic activity and were slightly more potent than our lead WC‐9. For example, two trifluoromethylated derivatives exhibited ED₅₀ values of 10.0 and 9.2 μM against intracellular T. cruzi, whereas they showed potent action against tachyzoites of T. gondii (ED₅₀ values of 1.6 and 1.9 μM against T. gondii). In addition, analogues of WC‐9 in which the terminal aryl group is in the meta position with respect to the alkyl chain bearing the thiocyanate group showed potent inhibitory action against both T. cruzi and T. gondii at the very low micromolar range, which suggests that a para‐phenyl substitution pattern is not necessary for biological activity.     

Investigation into the Feasibility of Thioditaloside as a Novel Scaffold for Galectin‐3‐Specific Inhibitors

Galectin‐3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this β‐galactose‐binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin‐1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour‐challenged mice. A novel approach—replacing galactose with its C2 epimer, talose—offers an alternative framework, as extensions at C2 permit exploitation of a galectin‐3‐specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin‐3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin‐3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.     

Structure‐Based Design of New KSP‐Eg5 Inhibitors Assisted by a Targeted Multicomponent Reaction

An integrated multidisciplinary approach that combined structure‐based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi+Boc removal/cyclization reaction sequence generated low‐micromolar‐range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds— 20 (IC₅₀=1.49 μM , EC₅₀=3.63 μM ) and 22 (IC₅₀=1.37 μM , EC₅₀=6.90 μM )—were synthesized with high efficiency by taking advantage of the multicomponent reactions.     

Chemical Genetics Approach to Engineer Kinesins with Sensitivity towards a Small‐Molecule Inhibitor of Eg5

Due to their fast and often reversible mode of action, small molecules are ideally suited to dissect biological processes. Yet, the validity of small‐molecule studies is intimately tied to the specificity of the applied compounds, thus imposing a great challenge to screens for novel inhibitors. Here, we applied a chemical‐genetics approach to render kinesin motor proteins sensitive to inhibition by the well‐characterized small molecule S‐Trityl‐l ‐cysteine (STLC). STLC specifically inhibits the kinesin Eg5 through binding to a known allosteric site within the motor domain. Transfer of this allosteric binding site into the motor domain of the human kinesins Kif3A and Kif4A sensitizes them towards STLC. Single‐molecule microscopy analyses confirmed that STLC inhibits the movement of chimeric but not wild‐type Kif4A along microtubules. Thus, our proof‐of‐concept study revealed that this chemical‐genetic approach provides a powerful strategy to specifically inhibit kinesins in vitro for which small‐molecule inhibitors are not yet available.     

Studies of Benzamide‐ and Thiol‐Based Histone Deacetylase Inhibitors in Models of Oxidative‐Stress‐Induced Neuronal Death: Identification of Some HDAC3‐Selective Inhibitors

Less stress : We compare three structurally different classes of histone deacetylase (HDAC) inhibitors that contain benzamide, hydroxamate, or thiol groups as the zinc binding group (ZBG) for their ability to protect cortical neurons in culture from cell death induced by oxidative stress. Novel benzamide‐based ligands selectively inhibit HDAC3 but provide no neuroprotection in the HCA–cortical neuron model of oxidative stress.

     

Synthesis and Biological Evaluation of Papain‐Family Cathepsin L‐Like Cysteine Protease Inhibitors Containing a 1,4‐Benzodiazepine Scaffold as Antiprotozoal Agents

Novel papain‐family cathepsin L‐like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure–activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3‐bromoisoxazoline moiety. Several rhodesain and falcipain‐2 inhibitors displaying single‐digit micromolar or sub‐micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.     

Indanones As High‐Potency Reversible Inhibitors of Monoamine Oxidase

Recent reports document that α‐tetralone (3,4‐dihydro‐2H‐naphthalen‐1‐one) is an appropriate scaffold for the design of high‐potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α‐tetralone and 1‐indanone, the present study involved synthesis of 34 1‐indanone and related indane derivatives as potential inhibitors of recombinant human MAO‐A and MAO‐B. The results show that C6‐substituted indanones are particularly potent and selective MAO‐B inhibitors, with IC₅₀ values ranging from 0.001 to 0.030 μM . C5‐Substituted indanone and indane derivatives are comparatively weaker MAO‐B inhibitors. Although the 1‐indanone and indane derivatives are selective inhibitors of the MAO‐B isoform, a number of homologues are also potent MAO‐A inhibitors, with three homologues possessing IC₅₀ values <0.1 μM . Dialysis of enzyme–inhibitor mixtures further established a selected 1‐indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1‐indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson’s disease and depression.     

Activity‐Fed Translation (AFT) Assay: A New High‐Throughput Screening Strategy for Enzymes in Droplets

There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet‐based microfluidics. Here we describe an original strategy, activity‐fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high‐throughput screening projects.     

Comparison of Nile Red and Cell Size Analysis for High‐Throughput Lipid Estimation Within Oleaginous Yeast

With growing interest in oleaginous yeast as producers of future fuels and bulk chemicals, a robust, high‐throughput method for estimating lipid production is required. Although the lipophilic dye Nile red is frequently used to assay large samples of yeast and microalgae, inconsistent stain permeability between species and strains limits its effectiveness for some microorganisms. In this study, the oleaginous yeast Metschnikowia pulcherrima is used to develop a fluorescence‐free, cell‐size‐based image analysis method for estimating lipid production, which is then compared with an optimized Nile red method across several experimental scenarios. Cell size analysis (CSA) outperforms Nile red in all scenarios, correlating well with lipid extraction data when screening multiple strains, screening a subset of strains grown in different conditions, and tracking the lipid accumulation of a culture over time. Stain permeability is shown to vary significantly among the strains trialled, with lipid droplet size and cell wall thickness having a deleterious effect in the permeability of high‐lipid‐accumulating cells. CSA can also allow culture population dynamics to be monitored, providing key process information of cell size distribution in response to changing media compositions. Practical Applications: Nile red is currently the go‐to method for high‐ throughput lipid screening; however, staining inconsistencies in some organisms caused by varying cell morphology makes it challenging to optimize a robust protocol. Although fluorescence‐free methods exist (Raman spectroscopy, Fourier‐transform infrared spectroscopy, GCMS), the need for extensive sample preparation and specialist equipment restricts their widespread adoption. The CSA method presented here offers an accurate, robust, and cheap alternative for the study of microorganisms where fluorescence‐based avenues are not feasible. Furthermore, the population dynamics collected during CSA can easily be applied to bioreactor style processing, where tracking size distributions can provide real time information of culture status. This additional information is valuable even if fluorescence screening is a possibility.     

In Vivo Efficacy of Natural Product‐Inspired Irreversible Kinase Inhibitors

Hypothemycin and related resorcylic acid lactones (RAL) bearing a cis‐enone moiety have emerged as an alternative pharmacophore to heterocyclic motifs for kinase inhibition, and are endowed with a unique selectivity filter based on the irreversible reaction with a subset of the kinome bearing a suitably positioned cysteine residue. Two prototypical examples of “edited” RAL were evaluated for antitumoral, antimetastatic and antiangiogenic efficacy in an orthotopic murine renal cell carcinoma (RENCA) model. Both compounds ( 3 and 5 ) are good inhibitors of VEGFRs in vitro, and inhibited tumor growth in vivo with comparable efficacy to sunitinib, an FDA‐approved VEGFRs inhibitor. Compound 3 promoted lung metastasis to a similar extent as sunitinib, while compound 5 strongly inhibited lung metastasis. This study attests to the potential of irreversible kinase inhibitors and molecular editing of natural pharmacophores and provides encouraging results to a clinically significant problem.     

The Lab Oddity Prevails: Discovery of Pan‐CDK Inhibitor (R)‐S‐Cyclopropyl‐S‐(4‐{[4‐{[(1R,2R)‐2‐hydroxy‐1‐methylpropyl]oxy}‐5‐(trifluoromethyl)pyrimidin‐2‐yl]amino}phenyl)sulfoximide (BAY 1000394) for the Treatment of Cancer

Lead optimization of a high‐throughput screening hit led to the rapid identification of aminopyrimidine ZK 304709, a multitargeted CDK and VEGF‐R inhibitor that displayed a promising preclinical profile. Nevertheless, ZK 304709 failed in phase I studies due to dose‐limited absorption and high inter‐patient variability, which was attributed to limited aqueous solubility and off‐target activity against carbonic anhydrases. Further lead optimization efforts to address the off‐target activity profile finally resulted in the introduction of a sulfoximine group, which is still a rather unusual approach in medicinal chemistry. However, the sulfoximine series of compounds quickly revealed very interesting properties, culminating in the identification of the nanomolar pan‐CDK inhibitor BAY 1000394, which is currently being investigated in phase I clinical trials.     

Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes

Although activity‐based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity‐based imaging of rhomboid proteases in membrane environments.