Blocked Inverted Indices for Exact Clustering of Large Chemical Spaces

The calculation of pairwise compound similarities based on fingerprints is one of the fundamental tasks in chemoinformatics. Methods for efficient calculation of compound similarities are of the utmost importance for various applications like similarity searching or library clustering. With the increasing size of public compound databases, exact clustering of these databases is desirable, but often computationally prohibitively expensive. We present an optimized inverted index algorithm for the calculation of all pairwise similarities on 2D fingerprints of a given data set. In contrast to other algorithms, it neither requires GPU computing nor yields a stochastic approximation of the clustering. The algorithm has been designed to work well with multicore architectures and shows excellent parallel speedup. As an application example of this algorithm, we implemented a deterministic clustering application, which has been designed to decompose virtual libraries comprising tens of millions of compounds in a short time on current hardware. Our results show that our implementation achieves more than 400 million Tanimoto similarity calculations per second on a common desktop CPU. Deterministic clustering of the available chemical space thus can be done on modern multicore machines within a few days

Identification of Two Secondary Ligand Binding Sites in 14-3‑3 Proteins Using Fragment Screening

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin <i>N</i>-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation

Inhibition of 14-3-3/Tau by Hybrid Small-Molecule Peptides Operating via Two Different Binding Modes

Current molecular hypotheses have not yet delivered marketable treatments for Alzheimer’s disease (AD), arguably due to a lack of understanding of AD biology and an overreliance on conventional drug modalities. Protein–protein interactions (PPIs) are emerging drug targets, which show promise for the treatment of, e.g., cancer, but are still underexploited for treating neurodegenerative diseases. 14-3-3 binding to phosphorylated Tau is a promising PPI drug target based on its reported destabilizing effect on microtubules, leading to enhanced neurofibrillary tangle formation as a potential cause of AD-related neurodegeneration. Inhibition of 14-3-3/Tau may therefore be neuroprotective. Previously, we reported the structure-guided development of modified peptide inhibitors of 14-3-3/Tau. Here, we report further efforts to optimize the binding mode and activity of our modified Tau peptides through a combination of chemical synthesis, biochemical assays, and X-ray crystallography. Most notably, we were able to characterize two different high-affinity binding modes, both of which inhibited 14-3-3-binding to full-length PKA-phosphorylated Tau protein in vitro as measured by NMR spectroscopy. Our findings, besides producing useful tool inhibitor compounds for studying 14-3-3/Tau, have enhanced our understanding of the molecular parameters for inhibiting 14-3-3/Tau, which are important milestones toward the establishment of our 14-3-3 PPI hypothesis

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein–Protein Interface

Protein regions that are involved in protein–protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein–protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular “Janus” ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein–protein interface, “freezes” one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein

Stabilization of Physical RAF/14-3‑3 Interaction by Cotylenin A as Treatment Strategy for RAS Mutant Cancers

One-third of all human cancers harbor somatic <i>RAS</i> mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF^V600 mutant melanomas. However, they paradoxically promote the growth of <i>RAS</i> mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in <i>RAS</i> mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth <i>in vitro</i> and <i>in vivo</i>. This defines a novel pharmacologic strategy for treatment of <i>RAS</i> mutant cancers

A Systematic Approach to the Discovery of Protein–Protein Interaction Stabilizers

Dysregulation of protein–protein interactions (PPIs) commonly leads to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for identifying reversibly covalent small molecules. We explored the scope of disulfide tethering for the discovery of selective PPI stabilizers (molecular glues) using the hub protein 14-3-3σ. We screened complexes of 14-3-3 with 5 biologically and structurally diverse phosphopeptides derived from the 14-3-3 client proteins ERα, FOXO1, C-RAF, USP8, and SOS1. Stabilizing fragments were found for 4/5 client complexes. Structural elucidation of these complexes revealed the ability of some peptides to conformationally adapt to make productive interactions with the tethered fragments. We validated eight fragment stabilizers, six of which showed selectivity for one phosphopeptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious fragment increased 14-3-3σ/C-RAF phosphopeptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3σ provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues

Therapeutic Opportunities Offered by the Excessive Lactate Production in Cancer

The majority of cancers of various tissue origin display wide portions suffering from insufficient respiration, due to permanent or transient hypoxia, which occurs during tumor development. This condition leads to the development of a glycolytic phenotype, where a compensatory lactate production takes place, in order to provide the cancer cells with sufficient amounts of energy and anabolites. Lactate is not just as a waste product of the glycolytic process, instead it plays a key role in the progression of cancer, since it promotes angiogenesis, cell migration, immune escape and radioresistance. Moreover, lactate can still constitute a metabolic fuel for oxidative tumor cells or vascular endothelial cells, and it can establish a symbiotic cell-cell shuttling system with stromal cells. Therefore, these peculiar roles of lactate in invasive tumors constitutes a high-priority target for future anti-cancer therapeutics [1]. Therapeutic interventions aimed at reducing lactate production in cancer tissues may consist of: a) reduction of glucose uptake (calorie-restricted ketogenic diet, physical exercise, inhibitors of glucose transporters); b) inhibition of enzymes involved in key-steps of glycolysis (inhibitors of hexokinase, phosphofructokinase, lactate dehydrogenase); c) block of the cellular trafficking of lactate (inhibitors of monocarboxylate transporters); d) enhancement of the mitochondrial oxidative metabolism (hyperbaric oxygen therapy, removal of inhibition of the Krebs cycle, for example, by using inhibitors of pyruvate dehydrogenase kinase) [2]. We have developed compounds that exert an anti-proliferative action on cancer cells by specific interventions on cancer metabolism, such as, inhibition of lactate dehydrogenase (LDH) activity [3,4], or reduction of glucose uptake through specific transmembrane transporters (GLUT) [5]. Furthermore, some of the LDH-inhibitors demonstrated a remarkable synergism with gemcitabine against pancreatic cancer cells in hypoxia [6]. and an improved activation of the redox-sensitive anti-cancer prodrug EO9 by means of an induced increase of the NADH/NAD+ cell ratio [7]. It is important to note that the development of agents that target lactate production, trafficking, and metabolism (by these or other methods) hold promise for treating nearly all invasive cancers, provided they present an appropriate therapeutic window. References 1) J. R. Doherty, J. L. Cleveland. J. Clin. Invest. 2013, 123, 3685–3692. 2) C. Granchi, F. Minutolo. ChemMedChem 2012, 7, 1318-1350. 3) C. Granchi, S. Roy, C. Giacomelli, et al. J. Med. Chem. 2011, 54, 1599–1612. 4) E. C. Calvaresi, C. Granchi, T. Tuccinardi, et al. ChemBioChem 2013, 14, 2263–2267. 5) T. Tuccinardi, C. Granchi, J. Iegre, et al. Bioorg. Med. Chem. Lett. 2013, 23, 6923–6927. 6) M. Maftouh, A. Avan, R. Sciarrillo, et al. Br. J. Cancer 2014, 110, 172-182. 7) S. J. Allison, J. R. P. Knight, C. Granchi, et al. Oncogenesis 2014, 3, e102; DOI: 10.1038/oncsis.2014.16

Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding

Designing Selective Drug-like Molecular Glues for the Glucocorticoid Receptor/14-3‑3 Protein–Protein Interaction

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein–protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators

Designing Selective Drug-like Molecular Glues for the Glucocorticoid Receptor/14-3‑3 Protein–Protein Interaction

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein–protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators