Identification of Novel Potent NSD2-PWWP1 Ligands Using Structure-Based Design and Computational Approaches.

Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.

Structural basis of the effect of activating mutations on the EGF receptor.

Mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are common oncogenic driver events in non-small cell lung cancer. Although the activation of EGFR in normal cells is primarily driven by growth-factor-binding-induced dimerization, mutations on different exons of the kinase domain of the receptor have been found to affect the equilibrium between its active and inactive conformations giving rise to growth-factor-independent kinase activation. Using molecular dynamics simulations combined with enhanced sampling techniques, we compare here the conformational landscape of the monomers and homodimers of the wild-type and mutated forms of EGFR ΔELREA and L858R, as well as of two exon 20 insertions, D770-N771insNPG, and A763-Y764insFQEA. The differences in the conformational energy landscapes are consistent with multiple mechanisms of action including the regulation of the hinge motion, the stabilization of the dimeric interface, and local unfolding transitions. Overall, a combination of different effects is caused by the mutations and leads to the observed aberrant signaling.

Structure-Activity Relationships of Hexahydrocyclopenta[c]quinoline Derivatives as Allosteric Inhibitors of CDK2 and EGFR.

Following the discovery of a type III allosteric modulator of cyclin-dependent kinase 2 (CDK2) characterized by a hexahydrocyclopenta[c]quinolone scaffold, three different series of its derivatives were synthesized and biologically evaluated. Docking of the synthesized compounds into the allosteric pocket of CDK2 allowed the elucidation of structure-activity relationships (SARs). Moreover, the compounds were tested on the wild-type epidermal growth factor receptor (EGFR) kinase domain (KD) and its clinically relevant T790M/L858R mutant form. Herein we describe the first SAR investigation of allosteric ligands that bind to the type III inhibitor pocket of CDK2 and EGFR-KD. Although the activity of the synthesized inhibitors needs to be improved, the obtained results provide clear-cut indications about pharmacophore requirements and selectivity determinants. Remarkably, this study led to the identification of a selective T790M/L858R EGFR allosteric inhibitor that is inactive toward both wild-type EGFR and CDK2. Finally, docking into the T790M/L858R EGFR-KD led us to hypothesize that the compounds bind to the double-mutant EGFR-KD by adopting a binding mode different from that in CDK2, thus rationalizing the observed selectivity profile.

Probing an Allosteric Pocket of CDK2 with Small Molecules.

The availability of well-characterized allosteric modulators is crucial for investigating the allosteric regulation of protein function. In a recently identified inactive conformation of cyclin-dependent kinase 2 (CDK2), an open allosteric pocket was detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Herein we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from that previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were found to be superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by crystallography. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. The activity of the obtained compounds was evaluated with various biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of lung (A549) and ovarian (SKOV3) cancer cell lines. Therefore, this report presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.

Identification and Structure-Activity Relationship Studies of Small-Molecule Inhibitors of the Methyllysine Reader Protein Spindlin1.

The methyllysine reader protein Spindlin1 has been implicated in the tumorigenesis of several types of cancer and may be an attractive novel therapeutic target. Small-molecule inhibitors of Spindlin1 should be valuable as chemical probes as well as potential new therapeutics. We applied an iterative virtual screening campaign, encompassing structure- and ligand-based approaches, to identify potential Spindlin1 inhibitors from databases of commercially available compounds. Our in silico studies coupled with in vitro testing were successful in identifying novel Spindlin1 inhibitors. Several 4-aminoquinazoline and quinazolinethione derivatives were among the active hit compounds, which indicated that these scaffolds represent promising lead structures for the development of Spindlin1 inhibitors. Subsequent lead optimization studies were hence carried out, and numerous derivatives of both lead scaffolds were synthesized. This resulted in the discovery of novel inhibitors of Spindlin1 and helped explore the structure-activity relationships of these inhibitor series.

Dual Kinase-Bromodomain Inhibitors in Anticancer Drug Discovery: A Structural and Pharmacological Perspective.

Protein kinases play crucial roles in several cell transformation processes and are validated drug targets for many human diseases, including cancer. Nevertheless, most tumors have eluded the effects of inhibition of a single kinase by activating resistance mechanisms and/or alternative pathways and escape mechanisms. In recent years, multitarget approaches directed toward inhibition of kinases and targets of different families have received increasing attention. In particular, co-targeting kinases and bromodomain epigenetic reader proteins has rapidly emerged as a promising approach to cancer drug development. In this manuscript, we will review the recent discoveries that led to the identification and optimization of dual kinase/bromodomain inhibitors. We will analyze and compare the structural features required for dual inhibition and comment on the potential of this approach in anticancer drug discovery. Moreover, we will introduce computational approaches useful for the identification of dual kinase/bromodomain inhibitors and generate ad hoc pharmacophore and docking models.

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2.

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

Nonpeptidic propargylamines as inhibitors of lysine specific demethylase 1 (LSD1) with cellular activity.

Lysine demethylases play an important role in epigenetic regulation and thus in the development of diseases like cancer or neurodegenerative disorders. As the lysine specific demethylase 1 (LSD1/KDM1) has been strongly connected to androgen and estrogen dependent gene expression, it serves as a promising target for the therapy of hormone dependent cancer. Here, we report on the discovery of new small molecule inhibitors of LSD1 containing a propargylamine warhead, starting out from lysine containing substrate analogues. On the basis of these substrate mimicking inhibitors, we were able to increase potency by a combination of similarity-based virtual screening and subsequent synthetic optimization resulting in more druglike LSD1 inhibitors that led to histone hypermethylation in breast cancer cells.

Synthesis, biological activity and molecular modeling studies on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors.

A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor activities were evaluated in vitro by using Ellman's method. It was discovered that most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selective against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were also carried out.

The role of histone demethylases in cancer therapy.

Reversible histone methylation has emerged in the last few years as an important mechanism of epigenetic regulation. Histone methyltransferases and demethylases have been identified as contributing factors in the development of several diseases, especially cancer. Therefore, they have been postulated to be new drug targets with high therapeutic potential. Here, we review histone demethylases with a special focus on their potential role in oncology drug discovery. We present an overview over the different classes of enzymes, their biochemistry, selected data on their role in physiology and already available inhibitors.

Computer- and structure-based lead design for epigenetic targets.

The term epigenetics is defined as inheritable changes that influence the outcome of a phenotype without changes in the genome. Epigenetics is based upon DNA methylation and posttranslational histone modifications. While there is much known about reversible acetylation as a posttranslational modification, research on reversible histone methylation is still emerging, especially with regard to drug discovery. As aberrant epigenetic modifications have been linked to many diseases, inhibitors of histone modifying enzymes are very much in demand. This article will summarize the progress on small molecule epigenetic inhibitors identified by structure- and computer-based approaches.