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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.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator binding sites within the GR ligand-binding domain that is crucial for the control of the functional response. However, the molecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD) simulations, bioinformatic analysis, and biophysical measurements are integrated to capture the structural and dynamic features of the allosteric cross-talk within the GR. We identified a network of evolutionarily conserved residues that enables the allosteric signal transduction, in agreement with experimental data. MD simulations clarify how such a network is dynamically interconnected and offer a mechanistic explanation of how different peptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GR allosteric regulation, ultimately providing a foundation for designing novel drugs.

Glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating thetranscription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator bindingsites within the GR ligand binding domain that is crucial for the control of the functional response. However, themolecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD)simulations, bioinformatic analysis and biophysical measurements are integrated to capture the structural anddynamic features of the allosteric cross-talk within GR. We identified a network of evolutionarily conservedresidues that enables the allosteric signal transduction, in agreement with experimental data. MD simulationsclarify how such network is dynamically interconnected and offer a mechanistic explanation of how the differentpeptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GRallosteric regulation, ultimately, posing the foundation for designing novel drugs.

Summary Aberrant expression ratio of Cl− transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clinical trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl− transporter NKCC2, creating critical drug compliance issues and health concerns. Here, we report the discovery of a new chemical class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiological intracellular Cl− in murine Down syndrome neuronal cultures, has excellent solubility and metabolic stability, and displays no issues with off-target activity in vitro. ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclinical/manufacturing studies toward the first sustainable therapeutics for the neurological conditions characterized by impaired Cl− homeostasis., Graphical Abstract, Highlights • NKCC1 is a promising target for the treatment of brain disorders • The newly discovered ARN23746 presents selective NKCC1 versus NKCC2 and KCC2 inhibition • ARN23746 restores altered neuronal chloride homeostasis in vitro • ARN23746 rescues core behaviors in DS and ASD mice with no diuretic effect or toxicity, The Bigger Picture In the last few decades, drug development for brain disorders has struggled to deliver effective small molecules as novel breakthrough classes of drugs. Discovery of effective chemical compounds for brain disorders has been greatly hampered by the fact that the few currently clinically used drugs were identified by serendipity, and these drugs’ mechanism of action is often poorly understood. Here, by leveraging drug repurposing as a means to quickly and safely evaluate the new pharmacological target NKCC1 and its implications in brain disorders in animal models and patients, we report an integrated strategy for the rational design and discovery of a novel, selective, and safe NKCC1 inhibitor, active in vivo. This compound has the potential to become a clinical drug candidate to treat several neurological conditions in patients. Eventually, this integrated drug-discovery strategy has the prospective to revive the appeal of drug-discovery programs in the challenging field of neuroscience., Currently, therapeutic options for several neurological disorders are scant or not highly effective. This is possibly due to the poor understanding of the mechanisms underlying these conditions. Here, starting from former validation of the new pharmacological target NKCC1 in brain disorders, we developed a novel, potent, and safe NKCC1 inhibitor, able to restore core behaviors in Down syndrome and autistic mouse models. This compound has the potential to become a solid drug candidate for the treatment of several neurological conditions.

Glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating thetranscription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator bindingsites within the GR ligand binding domain that is crucial for the control of the functional response. However, themolecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD)simulations, bioinformatic analysis and biophysical measurements are integrated to capture the structural anddynamic features of the allosteric cross-talk within GR. We identified a network of evolutionarily conservedresidues that enables the allosteric signal transduction, in agreement with experimental data. MD simulationsclarify how such network is dynamically interconnected and offer a mechanistic explanation of how the differentpeptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GRallosteric regulation, ultimately, posing the foundation for designing novel drugs.

The CDC42 family of GTPases (RHOJ, RHOQ, CDC42) control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. While recent studies have developed small molecules that target RAS GTPases, little progress has been made in targeting the related CDC42 family of GTPases for cancer treatment. Here, we use computer-aided drug design to identify a novel class of inhibitors that act on an allosteric pocket in the active form of the CDC42 GTPase, RHOJ. These allosteric inhibitors prevent RHOJ and CDC42 from binding to their downstream effector PAK, while having no effect on the interactions between the closely related GTPase RAC1 and PAK or RAS and its downstream effector RAF. Our lead compound ARN22089 has a druglike profile and can block both tumor growth and tumor angiogenesis in a three-dimensional vascularized microtumor (VMT) model, indicating that ARN22089 blocks RHOJ/CDC42 signaling in both the tumor cell and the tumor endothelium. Short term treatment of nascent melanoma tumors with ARN22089 halted the growth of BRAF mutant autochthonous mouse melanoma tumors, slowed the growth of melanoma patient-derived xenografts, and induced tumor necrosis in PDX models. In summary, we describe a multidisciplinary structure-based drug discovery platform that can identify new RHO family allosteric inhibitors and use this system to identify RHOJ inhibitors that block tumor growth in vivo. Citation Format: Jessica L. Flesher, Sohail Jahid, Jose A. Ortega, Giuseppina La Sala, Nicoletta Brindani, Jose M. Arencibia, Jacopo Manigrasso, Stephanie Hachey, Chi-Fen Chen, Chris Hughes, Marco De Vivo, Anand K. Ganesan. Structure-based design of CDC42/RHOJ effector inhibitors for the treatment of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5324.

Giuseppina La Sala earned her master’s degree in medicinal chemistry at Universita’ degli Studi di Siena in 2013. Then, she started her PhD in computational chemistry at Istituto Italiano di Tecnologia in Genoa under the supervision of Dr. Marco De Vivo. After her PhD, she worked as an application scientist in BiKi Technologies, a startup that develops methods based on molecular dynamics simulations and related approaches for drug design. In 2019, she joined AstraZeneca as an industrial postdoctoral fellow. Her research focuses on the study of protein-ligand interactions and the investigations of allosteric mechanisms in proteins.

Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abnormally high in melanoma cells. AC inhibition may thus be key to treating malignant melanoma. Here, we have used a systematic scaffold exploration to design a general pharmacophore for AC inhibition. This pharmacophore comprises a 6 + 5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are highly potent AC inhibitors. Their chemical and metabolic stabilities are comparable or superior to those of previously reported AC inhibitors. Moreover, they are potent against endogenous AC in intact melanoma cells. These novel inhibitors merit further characterization and can serve as a promising starting point for the discovery of new antimelanoma therapeutics.

The detection and characterization of binding pockets and allosteric communication in proteins is crucial for studying biological regulation and performing drug design. Nowadays, ever-longer molecular dynamics (MD) simulations are routinely used to investigate the spatiotemporal evolution of proteins. Yet, there is no computational tool that can automatically detect all the pockets and potential allosteric communication networks along these extended MD simulations. Here, we use a novel and fully automated algorithm that examines pocket formation, dynamics, and allosteric communication embedded in microsecond-long MD simulations of three pharmaceutically relevant proteins, namely, PNP, A2A, and Abl kinase. This dynamic analysis uses pocket crosstalk, defined as the temporal exchange of atoms between adjacent pockets, along the MD trajectories as a fingerprint of hidden allosteric communication networks. Importantly, this study indicates that dynamic pocket crosstalk analysis provides new mechanistic understandings on allosteric communication networks, enriching the available experimental data. Thus, our results suggest the prospective use of this unprecedented dynamic analysis to characterize transient binding pockets for structure-based drug design., Allosteric communication is revealed via protein pocket crosstalk networks, obtained by a novel and fully automated algorithm that examines pockets’ spatiotemporal evolution from extended MD simulations.

Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines.

Reference EPFL-CONF-229812View record in Web of Science Record created on 2017-07-10, modified on 2017-07-10

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.