Modulating protein activity with small-molecules binding to cryptic pockets offers great opportunities to overcome hurdles in drug design. Cryptic sites are atypical binding sites in proteins that ...are closed in the absence of a stabilizing ligand and are thus inherently difficult to identify. Many studies have proposed methods to predict cryptic sites. However, a general approach to prospectively sample open conformations of these sites and to identify cryptic pockets in an unbiased manner suitable for structure-based drug design remains elusive. Here, we describe an all-atom, explicit cosolvent, molecular dynamics (MD) simulations-based workflow to sample the open states of cryptic sites and identify opened pockets, in a manner that does not require a priori knowledge about these sites. Furthermore, the workflow relies on a target-independent parametrization that only distinguishes between binding pockets for peptides or small molecules. We validated our approach on a diverse test set of seven proteins with crystallographically determined cryptic sites. The known cryptic sites were found among the three highest-ranked predicted cryptic sites, and an open site conformation was sampled and selected for most of the systems. Crystallographic ligand poses were well reproduced by docking into these identified open conformations for five of the systems. When the fully open state could not be reproduced, we were still able to predict the location of the cryptic site, or identify other cryptic sites that could be retrospectively validated with knowledge of the protein target. These characteristics render our approach valuable for investigating novel protein targets without any prior information.
Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators ...were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.
Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these ...molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer.
We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model.
We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA-based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did.
The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.
The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to ...endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as “dileucine-based,” fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals.
Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to ...hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.
We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.
Sensitivity to binimetinib and ribociclib was inversely related (
= -0.58,
= 0.009).
amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.
Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling.
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To remain competitive, companies must decide on new, desirable products. This can be achieved by integrating insights how customers use a product into the process of deciding on a new product. ...Currently, this process is primarily based on market research that can only reveal the intention of consumers. Through the digitization of products, companies have access to large amounts of customer data that allow the application of data analytics methods. We provide a taxonomy of artificial intelligence, machine learning and data analysis, so that the notion of data analytics can be defined. Thus, the terms customer usage data, as well as a generic, five-stage product decision process (PDP) are defined and differentiated from consumer data and the product development process. Eventually, we show which data analytics methods on customer usage data can be used in order to tackle current challenges within the PDP. We incorporate the results of our structured literature review by connecting selected examples to our concept of the PDP. Our insights help to apply the proper data analytics methods in the PDP and thereby address the interplay between product decision and product development. Finally, future research directions for data analytics methods on customer usage data are put forward.
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•The notion of a product decision process is introduced.•A taxonomy for data analytics in the context of customer usage data is presented.•A literature review for data analytics methods to analyze usage data is provided.•A framework for data analytics methods in a product decision process is derived.•Future research areas for data analytics in a product decision process are derived.
Allostery describes the functional coupling between sites in biomolecules. Recently, the role of changes in protein dynamics for allosteric communication has been highlighted. A quantitative and ...predictive description of allostery is fundamental for understanding biological processes. Here, we integrate an ensemble-based perturbation approach with the analysis of biomolecular rigidity and flexibility to construct a model of dynamic allostery. Our model, by definition, excludes the possibility of conformational changes, evaluates static, not dynamic, properties of molecular systems, and describes allosteric effects due to ligand binding in terms of a novel free-energy measure. We validated our model on three distinct biomolecular systems: eglin c, protein tyrosine phosphatase 1B, and the lymphocyte function-associated antigen 1 domain. In all cases, it successfully identified key residues for signal transmission in very good agreement with the experiment. It correctly and quantitatively discriminated between positively or negatively cooperative effects for one of the systems. Our model should be a promising tool for the rational discovery of novel allosteric drugs.
The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in ...terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (K i < 100 nM) and measurable passive permeability (P app > 5 × 10–6 cm/s). Moreover, bioactive peptide 25 (K i = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.
The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) ...was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp3-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.
A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against ...HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.
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