Combining molecular dynamics and docking simulations to develop targeted protocols for performing optimized virtual screening campaigns on the HTRPM8 channel

Background: There is an increasing interest in TRPM8 ligands of medicinal interest, the rational design of which can be nowadays supported by structure-based in silico studies based on the recently resolved TRPM8 structures. Methods: The study involves the generation of a reliable hTRPM8 homology model, the reliability of which was assessed by a 1.0 \u3bcs MD simulation which was also used to generate multiple receptor conformations for the following structure-based virtual screening (VS) campaigns; docking simulations utilized different programs and involved all monomers of the selected frames; the so computed docking scores were combined by consensus approaches based on the EFO algorithm. Results: The obtained models revealed very satisfactory performances; LiGen\u2122 provided the best results among the tested docking programs; the combination of docking results from the four monomers elicited a markedly beneficial effect on the computed consensus models. Conclusions: The generated hTRPM8 model appears to be amenable for successful structure-based VS studies; cross-talk modulating effects between interacting monomers on the binding sites can be accounted for by combining docking simulations as performed on all the monomers; this strategy can have general applicability for docking simulations involving quaternary protein structures with multiple identical binding pockets

Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand-and structure-based virtual screening approach

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38

Combining Different Docking Engines and Consensus Strategies to Design and Validate Optimized Virtual Screening Protocols for the SARS-CoV-2 3CL Protease

The 3CL-Protease appears to be a very promising medicinal target to develop anti-SARS-CoV-2 agents. The availability of resolved structures allows structure-based computational approaches to be carried out even though the lack of known inhibitors prevents a proper validation of the performed simulations. The innovative idea of the study is to exploit known inhibitors of SARS-CoV 3CL-Pro as a training set to perform and validate multiple virtual screening campaigns. Docking simulations using four different programs (Fred, Glide, LiGen, and PLANTS) were performed investigating the role of both multiple binding modes (by binding space) and multiple isomers/states (by developing the corresponding isomeric space). The computed docking scores were used to develop consensus models, which allow an in-depth comparison of the resulting performances. On average, the reached performances revealed the different sensitivity to isomeric differences and multiple binding modes between the four docking engines. In detail, Glide and LiGen are the tools that best benefit from isomeric and binding space, respectively, while Fred is the most insensitive program. The obtained results emphasize the fruitful role of combining various docking tools to optimize the predictive performances. Taken together, the performed simulations allowed the rational development of highly performing virtual screening workflows, which could be further optimized by considering different 3CL-Pro structures and, more importantly, by including true SARS-CoV-2 3CL-Pro inhibitors (as learning set) when available

Production of human α-mannosidase in plant

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy

Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2

Chemokines CXCL8 and CXCL1 play a key role in the recruitment of neutrophils at the site of inflammation. CXCL8 binds two membrane receptors, CXCR1 and CXCR2, whereas CXCL1 is a selective agonist for CXCR2. In the past decade, the physiopathological role of CXCL8 and CXCL1 has been investigated. A novel class of small molecular weight allosteric CXCR1 inhibitors was identified, and reparixin, the first drug candidate, is currently under clinical investigation in the prevention of ischemia/reperfusion injury in organ transplantation. Reparixin binding mode to CXCR1 has been studied and used for a computer-assisted design program of dual allosteric CXCR1 and CXCR2 inhibitors. In this paper, the results of modeling-driven SAR studies for the identification of potent dual inhibitors are discussed, and three new compounds (56, 67, and 79) sharing a common triflate moiety have been selected as potential leads with optimized pharmacokinetic characteristic

The ANTAREX tool flow for monitoring and autotuning energy efficient HPC systems

Designing and optimizing HPC applications are difficult and complex tasks, which require mastering specialized languages and tools for performance tuning. As this is incompatible with the current trend to open HPC infrastructures to a wider range of users, the availability of more sophisticated programming languages and tools to assist and automate the design stages is crucial to provide smoothly migration paths towards novel heterogeneous HPC platforms. The ANTAREX project intends to address these issues by providing a tool flow, a Domain Specific Launguage and APIs to provide application's adaptivity and to runtime manage and autotune applications for heterogeneous HPC systems. Our DSL provides a separation of concerns, where analysis, runtime adaptivity, performance tuning and energy strategies are specified separately from the application functionalities with the goal to increase productivity, significantly reduce time to solution, while making possible the deployment of substantially improved implementations. This paper presents the ANTAREX tool flow and shows the impact of optimization strategies in the context of one of the ANTAREX use cases related to personalized drug design. We show how simple strategies, not devised by typical compilers, can substantially speedup the execution and reduce energy consumption

The ANTAREX domain specific language for high performance computing

The ANTAREX project relies on a Domain Specific Language (DSL) based on Aspect Oriented Programming (AOP) concepts to allow applications to enforce extra functional properties such as energy-efficiency and performance and to optimize Quality of Service (QoS) in an adaptive way. The DSL approach allows the definition of energy-efficiency, performance, and adaptivity strategies as well as their enforcement at runtime through application autotuning and resource and power management. In this paper, we present an overview of the key outcome of the project, the ANTAREX DSL, and some of its capabilities through a number of examples, including how the DSL is applied in the context of the project use cases

Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction