Fluorine NMR functional screening: from purified enzymes to human intact living cells.

The substrate- or cofactor-based fluorine NMR screening, also known as n-FABS (n fluorine atoms for biochemical screening), represents a powerful method for performing a direct functional assay in the search of inhibitors or enhancers of an enzymatic reaction. Although it suffers from the intrinsic low sensitivity compared to other biophysical techniques usually applied in functional assays, it has some distinctive features that makes it appealing for tackling complex chemical and biological systems. Its strengths are represented by the easy set-up, robustness, flexibility, lack of signal interference and rich information content resulting in the identification of bona fide inhibitors and reliable determination of their inhibitory strength. The versatility of the n-FABS allows its application to either purified enzymes, cell lysates or intact living cells. The principles, along with theoretical, technical and practical aspects, of the methodology are discussed. Furthermore, several applications of the technique to pharmaceutical projects are presented.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Predicting polypharmacology by binding site similarity: from kinases to the protein universe.

Polypharmacology is receiving increasing attention in the pharmaceutical industry, since finding new targets of a compound is useful not only for anticipating possible side effects but also for opening new therapeutic opportunities. Thus, while system biology and personalized medicine are becoming increasingly important, there is an urgent need to map the inhibition profile of a compound on a large panel of targets by using both experimental and computational methods. This is especially important for kinase inhibitors, given the high similarity at the binding site level for the 518 kinases in the human genome. In this paper, we propose and validate a new method to predict the inhibition map of a compound by comparison of binding pockets. We used a subset of the Ambit panel for the validation-17 inhibitors with K(d) measured on 189 kinases-and found that on average 37% of kinases inhibited with K(d) < 10 microM were retrieved at 10% ROC enrichment. These results make this method particularly suitable to rationalize and optimize the selectivity profile of a compound. In addition, the method was extended to explore all the proteins in the PDB by using as queries pockets occupied by compounds of biological interest (ATP and various marketed drugs). The profiling of compounds against the protein universe revealed that striking structural similarities at the subpocket level (RMSD < 0.5 A) may also occur among targets with different folds, which can be exploited not only to predict off-target effects but also to design novel inhibitors for the target of interest.

Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors.

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.

Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor.

The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.

6-Substituted pyrrolo[3,4-c]pyrazoles: an improved class of CDK2 inhibitors.

We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.

Polyfluorinated amino acids for sensitive 19F NMR-based screening and kinetic measurements.

Two novel series of polyfluorinated amino acids (PFAs) were designed and synthesized according to a very short and scalable synthetic sequence. The advantages and limitations of these moieties for screening purposes are presented and discussed. The potential applications of these PFAs were tested with their incorporation into small arginine-containing peptides that represent suitable substrates for the enzyme trypsin. The enzymatic reactions were monitored by 19F NMR spectroscopy, using the 3-FABS (three fluorine atoms for biochemical screening) technique. The high sensitivity achieved with these PFAs permits a reduction in substrate concentration required for 3-FABS. This is relevant in the utilization of 3-FABS in fragment-based screening for identification of small scaffolds that bind weakly to the receptor of interest. The large dispersion of 19F isotropic chemical shifts allows the simultaneous measurement of the efficiency of the different substrates, thus identifying the best substrate for screening purposes. Furthermore, the knowledge of KM and Kcat for the different substrates allows the identification of the structural motifs responsible for the binding affinity to the receptor and those affecting the chemical steps in enzymatic catalysis. This enables the construction of suitable pharmacophores that can be used for designing nonpeptidic inhibitors with high affinity for the enzyme or molecules that mimic the transition state. The novel PFAs can also find useful application in the FAXS (fluorine chemical shift anisotropy and exchange for screening) experiment, a 19F-based competition binding assay for the detection of molecules that inhibit the interaction between two proteins.

NMR-based quality control approach for the identification of false positives and false negatives in high throughput screening.

The quality of the data generated in a high throughput screening (HTS) run is fundamental for selecting bona fide inhibitors and for ensuring the capture of the full richness of inhibitors present in a chemical library. For this purpose a quality control filter based on three one dimensional (1D) proton NMR experiments is proposed. The approach called SPAM (Solubility, Purity and Aggregation of the Molecule) Filter requires the acquisition of a 1D reference spectrum, a WaterLOGSY spectrum and/or a selective longitudinal relaxation filter spectrum for the identified hits dissolved in aqueous solution and in the presence of a water soluble reference molecule. This palette of experiments permits the rapid characterization of the identity, purity, solubility and aggregation state of the active compound. This knowledge is crucial for deriving accurate IC(50) and K(1) values of the inhibitors, for identifying false negatives and for detecting promiscuous inhibitors. Only compounds that pass through the SPAM Filter can be considered as starting points for medicinal chemistry efforts directed toward lead optimization. Examples of this approach in the identification of false positives in a screening run against the enzyme thymidine phosphorylase (TP) and the rescue of a false negative in a screening run against the Ser/Thr kinase AKT1 are presented.

3-Amino-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: a new class of CDK2 inhibitors.

We have recently reported about a new class of Aurora-A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. Here we describe the synthesis and early expansion of CDK2/cyclin A-E inhibitors belonging to the same chemical class. Synthesis of the compounds was accomplished using a solution-phase protocol amenable to rapid parallel expansion. Compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit CDK2-mediated tumor cell proliferation have been obtained.

Structure-based approaches to improve selectivity: CDK2-GSK3beta binding site analysis.

An evaluation and comparison of two different approaches, GRID/CPCA and GRIND/CPCA (CPCA = consensus principal component analysis; GRIND = GRid-INdependent Descriptors), suitable for visualizing the structural differences between related proteins is presented. Ten crystal structures of CDK2/cyclin A and GSK3beta solved in-house with different inhibitors were compared with the aim of highlighting regions that could be potential sites for gaining selectivity for CDK2 versus GSK3beta. The analyses pointed out remarkable differences in the backs of the CDK2-GSK3beta ATP binding pockets that guided the optimization toward a selective benzodipyrazole CDK2 inhibitor. The gain in selectivity can be associated with the two main differences in the ATP pocket between the enzymes. Phe80 of CDK2, the so-called gatekeeper residue often exploited for the design of kinase selective ligands, is replaced by a leucine in GSK3beta, and Ala144 is replaced by a cysteine. As a consequence of these mutations, CDK2 has a less elongated and less flat buried region at the back of the ATP pocket.

An analysis of the binding modes of ATP-competitive CDK2 inhibitors as revealed by X-ray structures of protein-inhibitor complexes.

CDK2 is an attractive target for the design of new therapeutic antitumor agent. Numerous CDK2 inhibitors have been discovered and their crystallographic structures either in complex with CDK2 or CDK2/Cyclin A have been published. This review aims to summarize the publicly available structural characterization of CDK2/(Cyclin A) -- ligand complexes and to highlight the similarities among the binding modes of structurally diverse inhibitors.

Virtual screening to enrich a compound collection with CDK2 inhibitors using docking, scoring, and composite scoring models.

Docking programs can generate subsets of a compound collection with an increased percentage of actives against a target (enrichment) by predicting their binding mode (pose) and affinity (score), and retrieving those with the highest scores. Using the QXP and GOLD programs, we compared the ability of six single scoring functions (PLP, Ligscore, Ludi, Jain, ChemScore, PMF) and four composite scoring models (Mean Rank: MR, Rank-by-Vote: Vt, Bayesian Statistics: BS and PLS Discriminant Analysis: DA) to separate compounds that are active against CDK2 from inactives. We determined the enrichment for the entire set of actives (IC50 < 10 microM) and for three activity subsets. In all cases, the enrichment for each subset was lower than for the entire set of actives. QXP outperformed GOLD at pose prediction, but yielded only moderately better enrichments. Five to six scoring functions yielded good enrichments with GOLD poses, while typically only two worked well with QXP poses. For each program, two scoring functions generally performed better than the others (Ligscore2 and Ludi for GOLD; QXP and Jain for QXP). Composite scoring functions yielded better results than single scoring functions. The consensus approaches MR and Vt worked best when separating micromolar inhibitors from inactives. The statistical approaches BS and DA, which require training data, performed best when distinguishing between low and high nanomolar inhibitors. The key observation that all hit rate profiles for all four activity intervals for all scoring schemes for both programs are significantly better than random, is evidence that docking can be successfully applied to enrich compound collections.

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 2. Lead optimization.

Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K(i) of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC(50) in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.

Potent and selective Aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition.

Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent compound reported in this study had an IC(50) of 0.027 microM in the enzymatic assay for Aur-A inhibition and IC(50)s between 0.05 microM and 0.5 microM for the inhibition of proliferation of different tumor cell lines.

Benzodipyrazoles: a new class of potent CDK2 inhibitors.

The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible lead development. Following a medicinal chemistry program aimed at improving cell permeability and selectivity, a series of compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit tumor cell proliferation has been obtained.

Sequence and structural analysis of kinase ATP pocket residues.

Protein kinases represent one of the largest known families of enzymes. Most kinases bind ATP and most synthetic kinase inhibitors are ATP-competitive, which makes selectivity a potential problem. However, despite the high sequence similarity in the ATP binding pocket, several groups including ours have been able to develop highly potent and selective ATP-competitive inhibitors. To systematically aid the design of specific inhibitors in our protein kinase projects, we aligned all known three-dimensional structures and all known sequences of human protein kinases. We identified a set of 38 residues that make up the ATP pocket and analyzed the variability among these residues. The most variable residues in the ATP pocket are targeted to design specificity into inhibitors in our various kinase projects.

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 1. Lead finding.

Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.