Antagonism of the Stat3-Stat3 protein dimer with salicylic acid based small molecules

More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure–activity relationship (SAR) study based on the previously identified inhibitor S3I‐201 (IC₅₀=86 μM , K_i>300 μM ). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC₅₀ range of 18.7–51.9 μM , and disruption of Stat3–pTyr peptide interactions with K_i values in the 15.5–41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.     

A Highly Potent and Cellularly Active β‐Peptidic Inhibitor of the p53/hDM2 Interaction

New and improved : The incorporation of a 6‐chlorotryptophan (6‐Cl‐Trp) into a β‐peptide (M)‐3₁₄ helix leads to a high‐affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.

     

Rational Design,Binding Studies,and Crystal‐Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14‐3‐3ζ Adapter Protein

14‐3‐3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14‐3‐3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14‐3‐3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X‐ray crystallography.     

The Cell Surface Receptor CD44: NMR‐Based Characterization of Putative Ligands

The cell surface receptor CD44 is a glycoprotein belonging to the hyaluronan‐binding proteins, termed hyaladherins. CD44 is expressed in a wide variety of isoforms in many cells and, in particular, is present on the surface of malignant cells where it is involved in the onset and progression of cancer. In a first attempt to identify novel CD44‐binding agents, we first characterized, with NMR spectroscopic techniques, several agents that were reported to bind to human CD44 (hCD44). To our surprise, however, none of these putative CD44‐binding agents, including a peptide that is in phase 2 clinical trials (A6 peptide) and a recently reported fragment hit, were found to interact significantly with recombinant hCD44(21–178). Nonetheless, we further report that a fragment‐screening campaign, with solution NMR spectroscopy as the detection method, identified a viable fragment hit that bound in a potentially functional pocket on the surface of CD44, opposite to the hyaluronic acid binding site. We hypothesize that this pocket could be indirectly associated with the cellular and in vivo activity of the A6 peptide, which would provide a novel framework for the possible development of therapeutically viable CD44 antagonists.     

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

Cyclin‐dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small‐molecule inhibitors of CDK–CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S‐phase cell populations, and cause a decrease in CDK2, cyclin A, and p27^Kip1 levels. These molecules should help decipher the complex contributions of CDK–CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.     

Development of 3‐Phenyl‐N‐(2‐(3‐phenylureido)ethyl)‐thiophene‐2‐sulfonamide Compounds as Inhibitors of Antiapoptotic Bcl‐2 Family Proteins

Antiapoptotic Bcl‐2 family proteins, such as Bcl‐x_L, Bcl‐2, and Mcl‐1, are often overexpressed in tumor cells, which contributes to tumor cell resistance to chemotherapies and radiotherapies. Inhibitors of these proteins thus have potential applications in cancer treatment. We discovered, through structure‐based virtual screening, a lead compound with micromolar binding affinity to Mcl‐1 (inhibition constant (K_i)=3 μM ). It contains a phenyltetrazole and a hydrazinecarbothioamide moiety, and it represents a structural scaffold not observed among known Bcl‐2 inhibitors. This work presents the structural optimization of this lead compound. By following the scaffold‐hopping strategy, we have designed and synthesized a total of 82 compounds in three sets. All of the compounds were evaluated in a fluorescence‐polarization binding assay to measure their binding affinities to Bcl‐x_L, Bcl‐2, and Mcl‐1. Some of the compounds with a 3‐phenylthiophene‐2‐sulfonamide core moiety showed sub‐micromolar binding affinities to Mcl‐1 (K_i=0.3–0.4 μM ) or Bcl‐2 (K_i≈1 μM ). They also showed obvious cytotoxicity on tumor cells (IC₅₀<10 μM ). Two‐dimensional heteronuclear single quantum coherence NMR spectra of three selected compounds, that is, YCW‐E5, YCW‐E10, and YCW‐E11, indicated that they bind to the BH3‐binding groove on Bcl‐x_L in a similar mode to ABT‐737. Several apoptotic assays conducted on HL‐60 cells demonstrated that these compounds are able to induce cell apoptosis through the mitochondrial pathway. We propose that the compounds with the 3‐phenylthiophene‐2‐sulfonamide core moiety are worth further optimization as effective apoptosis inducers with an interesting selectivity towards Mcl‐1 and Bcl‐2.     

Biophysical Studies of the Amyloid β‐Peptide: Interactions with Metal Ions and Small Molecules

Alzheimer's disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.     

Lectin‐Based Drug Design: Combined Strategy to Identify Lead Compounds using STD NMR Spectroscopy,Solid‐Phase Assays and Cell Binding for a Plant Toxin Model

The growing awareness of the sugar code—i.e. the biological functionality of glycans—is leading to increased interest in lectins as drug targets. The aim of this study was to establish a strategic combination of screening procedures with increased biorelevance. As a model, we used a potent plant toxin (viscumin) and lactosides synthetically modified at the C6/C6′ positions and the reducing end aglycan. Changes in the saturation transfer difference (STD) in NMR spectroscopy, applied in inhibition assays, yielded evidence for ligand activity and affinity differences. Inhibitory potency was confirmed by the blocking of lectin binding to a glycoprotein‐bearing matrix. In cell‐based assays, iodo/azido‐substituted lactose derivatives were comparatively active. Interestingly, cell‐type dependence was observed, indicating the potential of synthetic carbohydrate derivative to interact with lectins in a cell‐type (glycan profile)‐specific manner. These results are relevent to research into human lectins, glycosciences, and beyond.     

Foldamers as Anticancer Therapeutics: Targeting Protein–Protein Interactions and the Cell Membrane

Targeting important protein–protein interactions involved in carcinogenesis or targeting the cell membrane of a cancer cell directly are just two of the ways in which foldamers (oligomeric molecules that fold into distinct shapes in solution) hold considerable potential in the treatment of cancer. From mimicking the local topography of the helical compound of interest by using covalently constrained foldamers to mimicking the topography of the natural helix such that the positions of key functional motifs are in an identical spatial orientation to match those presented by the original α‐helix, synthetic foldamers have been used to mimic the natural foldamers that interact with proteins or the cell membrane. These targeted approaches have become established over a timeframe of more than a decade, and they continue to be included in the assortment of cancer targets being studied and the arsenal of chemotherapy compounds in development. These approaches are reviewed herein.     

A Novel Prodrug of a γ‐Glutamylcyclotransferase Inhibitor Suppresses Cancer Cell Proliferation in vitro and Inhibits Tumor Growth in a Xenograft Mouse Model of Prostate Cancer

γ‐Glutamylcyclotransferase (GGCT) depletion inhibits cancer cell proliferation. However, whether the enzymatic activity of GGCT is critical for the regulation of cancer cell growth remains unclear. In this study, a novel diester‐type cell‐permeable prodrug, pro‐GA, was developed based on the structure of N‐glutaryl‐l ‐alanine (GA), by structure optimization using temporary fluorophore‐tagged prodrug candidates. The antiproliferative activity of pro‐GA was demonstrated using GGCT‐overexpressing NIH‐3T3 cells and human cancer cells including MCF7, HL‐60, and PC3 cells. By contrast, normal cells were not significantly affected by pro‐GA treatment. Moreover, pro‐GA administration exhibited anticancer effects in a xenograft model using immunocompromised mice inoculated with PC3 cells. These results indicate that the enzymatic activity of GGCT accelerates tumor growth and that GGCT inhibition is a promising therapeutic strategy for the treatment of GGCT‐overexpressing tumors.     

Activity‐Based Probes for HECT E3 Ubiquitin Ligases

Activity‐based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine‐based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin‐ABPs (Ub‐VME and Ub‐PA) and a novel set of E2–Ub‐ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin‐ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub‐ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub‐PA and His‐UBE2D2–Ub‐ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases.     

A Comprehensive Structural Overview of p38α Mitogen‐Activated Protein Kinase in Complex with ATP‐Site and Non‐ATP‐Site Binders

Herein we review all the currently available ATP‐site and non‐ATP‐site ligands bound to p38α mitogen‐activated protein kinase (MAPK) available in the RCSB Protein Data Bank (PDB). The co‐crystallized inhibitors have been classified into different families according to their experimental binding mode and chemical structure, and the ligand–protein interactions are discussed using the most representative compounds. This systematic structural analysis could provide some take‐home lessons for drug discovery programs aimed at the rational identification and optimization of new p38α MAPK inhibitors.     

Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.