Development of Optimal Virtual Screening Strategies to Identify Novel Toll-Like Receptor Ligands Using the DockBox Suite.

Toll-like receptors (TLRs) represent attractive targets for developing modulators for the treatment of many pathologies, including inflammation, cancer, and autoimmune diseases. Here, we describe a protocol based on the DockBox package that enables to set up and perform structure-based virtual screening in order to increase the chance of identifying novel TLR ligands from chemical libraries.

Linkers in fragment-based drug design: an overview of the literature.

INTRODUCTION: In fragment-based drug design, fragment linking is a popular strategy where two fragments binding to different sub-pockets of a target are linked together. This attractive method remains challenging especially due to the design of ideal linkers. AREAS COVERED: The authors review the types of linkers and chemical reactions commonly used to the synthesis of linkers, including those utilized in protein-templated fragment self-assembly, where fragments are directly linked in the presence of the protein. Finally, they detail computational workflows and software including generative models that have been developed for fragment linking. EXPERT OPINION: The authors believe that fragment linking offers key advantages for compound design, particularly for the design of bivalent inhibitors linking two distinct pockets of the same or different subunits. On the other hand, more studies are needed to increase the potential of protein-templated approaches in FBDD. Important computational tools such as structure-based de novo software are emerging to select suitable linkers. Fragment linking will undoubtedly benefit from developments in computational approaches and machine learning models.

Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules.

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.

A Deep Dive into VDAC1 Conformational Diversity Using All-Atom Simulations Provides New Insights into the Structural Origin of the Closed States.

The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter that controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high-conducting "open" state and a low-conducting "closed" state emerging at high transmembrane (TM) potentials. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here, we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltages known to promote closure. Conformers exhibiting durable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus, which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our models suggest that stable low-conducting states of VDAC1 predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed experimentally and corroborated a recent study describing entropy as a key factor for VDAC gating.

Combretastatin A-4 sulfur-containing heterocyclic derivatives: Synthesis, antiproliferative activities and molecular docking studies.

Combretastatin A-4 inspired heterocyclic derivatives were synthesized and evaluated for their biological activities on tubulin polymerization and cell proliferation. Among the 19 described sulfur-containing compounds, derivatives (Z)-4h and (Z)-4j exhibited interesting in cellulo tubulin polymerization inhibition and antiproliferative activities with IC50 values for six different cell lines between 8 and 27 nM. Furthermore, in silico docking studies within the colchicine/CA-4 binding site of tubulin were carried out to understand the interactions of our products with the protein target. The effects on the cell cycle of follicular lymphoma cells were also investigated at 1-10 nM concentrations showing that apoptotic processes occurred.

The intrinsically disordered N-terminus of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is a critical ß-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the ß-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.

Synthesis, anticancer activity and molecular docking studies of N-deacetylthiocolchicine and 4-iodo-N-deacetylthiocolchicine derivatives.

Colchicine is a plant alkaloid with a broad spectrum of biological and pharmacological properties. It has found application as an anti-inflammatory agent and also shows anticancer effects through its ability to destabilize microtubules by preventing tubulin dimers from polymerizing leading to mitotic death. However, adverse side effects have so far restricted its use in cancer therapy. This has led to renewed efforts to identify less toxic derivatives. In this article, we describe the synthesis of a set of novel double- and triple-modified colchicine derivatives. These derivatives were tested against primary acute lymphoblastic leukemia (ALL-5) cells and several established cancer cell lines including A549, MCF-7, LoVo and LoVo/DX. The novel derivatives were active in the low nanomolar range, with 7-deacetyl-10-thiocolchicine analogues more potent towards ALL-5 cells while 4-iodo-7-deacetyl-10-thiocolchicine analogues slightly more effective towards the LoVo cell line. Moreover, most of the synthesized compounds showed a favorable selectivity index (SI), particularly for ALL-5 and LoVo cell lines. Cell cycle analysis of the most potent molecules on ALL-5 and MCF-7 cell lines revealed contrasting effects, where M-phase arrest was observed in MCF-7 cells but not in ALL-5 cells. Molecular docking studies of all derivatives to the colchicine-binding site were performed and it was found that five of the derivatives showed strong ß-tubulin binding energies, lower than -8.70 kcal/mol, while the binding energy calculated for colchicine is -8.09 kcal/mol. The present results indicate that 7-deacetyl-10-thiocolchicine and 4-iodo-7-deacetyl-10-thiocolchicine analogues constitute promising lead compounds as chemotherapy agents against several types of cancer.

Fgd5 is a Rac1-specific Rho GEF that is selectively inhibited by aurintricarboxylic acid.

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators. Abbreviations: a.a.: amino acid; ATA: aurintricarboxylic acid; DH: Dbl homology; DOCK: dictator of cytokinesis; Fgd: faciogenital dysplasia; GEF: guanine-nucleotide exchange factor; GST: glutathione S-transferase; LOPAC: library of pharmacologically active compounds; PH: pleckstrin homology; PDB: protein data bank; s.e.m.: standard error of the mean; SPR: surface plasmon resonance.

Revealing and Attenuating the Electrostatic Properties of Tubulin and Its Polymers.

Tubulin is an electrostatically negative protein that forms cylindrical polymers termed microtubules, which are crucial for a variety of intracellular roles. Exploiting the electrostatic behavior of tubulin and microtubules within functional microfluidic and optoelectronic devices is limited due to the lack of understanding of tubulin behavior as a function of solvent composition. This work displays the tunability of tubulin surface charge using dimethyl sulfoxide (DMSO) for the first time. Increasing the DMSO volume fractions leads to the lowering of tubulin's negative surface charge, eventually causing it to become positive in solutions >80% DMSO. As determined by electrophoretic mobility measurements, this change in surface charge is directionally reversible, i.e., permitting control between -1.5 and + 0.2 cm2  (V s)-1 . When usually negative microtubules are exposed to these conditions, the positively charged tubulin forms tubulin sheets and aggregates, as revealed by an electrophoretic transport assay. Fluorescence-based experiments also indicate that tubulin sheets and aggregates colocalize with negatively charged g-C3 N4 sheets while microtubules do not, further verifying the presence of a positive surface charge. This study illustrates that tubulin and its polymers, in addition to being mechanically robust, are also electrically tunable.

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT-1: Influence on 18 F-FDG PET Uptake.

Positron emission tomography (PET) using 2-deoxy-2-[18 F]fluoro-d-glucose ([18 F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both nondiabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter-1 (hGLUT-1) in FaDu and GIST-1 cells by measuring [3 H]2-deoxy-d-glucose ([3 H]2-DG) and [3 H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [3 H]2-DG uptake. In GIST-1 cells, [3 H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H-pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT-1. We hypothesize that inhibition of hGLUT-1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [18 F]FDG uptake, increasingly used as a marker of tumor response. The hGLUT-1 inhibition by TKIs may have implications for routine [18 F]FDG-PET monitoring of tumor response in patients.

Comparative Analysis, Structural Insights, and Substrate/Drug Interaction of CYP128A1 in Mycobacterium tuberculosis.

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms' primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family's distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1, or together with CYP124A1 and CYP121A1. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis.

Synthesis, biological evaluation and molecular docking studies of new amides of 4-chlorothiocolchicine as anticancer agents.

Colchicine belongs to a large group of microtubule polymerization inhibitors. Although the anti-cancer activity of colchicine and its derivatives has been established, none of them has found commercial application in cancer treatment due to side effects. Therefore, we designed and synthesized a series of six triple-modified 4-chlorothiocolchicine analogues with amide moieties and one urea derivative. These novel derivatives were tested against several different cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) and primary acute lymphoblastic leukemia (ALL) cells and they showed activity in the nanomolar range. The obtained IC50 values for novel derivatives were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies of colchicine and selected analogues were undertaken to indicate that they induced apoptotic cell death in ALL-5 cells. We also performed in silico studies to predict binding modes of the 4-chlorothiocolchicine derivatives to different ß tubulin isotypes. The results indicate that select triple-modified 4-chlorothiocolchicine derivatives represent highly promising novel cancer chemotherapeutics.

Investigation of the Electrical Properties of Microtubule Ensembles under Cell-Like Conditions.

Microtubules are hollow cylindrical polymers composed of the highly negatively-charged (~23e), high dipole moment (1750 D) protein α, ß- tubulin. While the roles of microtubules in chromosomal segregation, macromolecular transport, and cell migration are relatively well-understood, studies on the electrical properties of microtubules have only recently gained strong interest. Here, we show that while microtubules at physiological concentrations increase solution capacitance, free tubulin has no appreciable effect. Further, we observed a decrease in electrical resistance of solution, with charge transport peaking between 20-60 Hz in the presence of microtubules, consistent with recent findings that microtubules exhibit electric oscillations at such low frequencies. We were able to quantify the capacitance and resistance of the microtubules (MT) network at physiological tubulin concentrations to be 1.27 × 10-5 F and 9.74 × 104 Ω. Our results show that in addition to macromolecular transport, microtubules also act as charge storage devices through counterionic condensation across a broad frequency spectrum. We conclude with a hypothesis of an electrically tunable cytoskeleton where the dielectric properties of tubulin are polymerisation-state dependent.

Synthesis, antiproliferative activity, and molecular docking studies of 4-chlorothiocolchicine analogues.

Colchicine is a therapeutic agent currently used in therapies of many diseases. It also shows antimitotic effects, and its high cytotoxic activity against different cancer cell lines has been demonstrated many times. To overcome the limitations of colchicine use in anticancer therapy, we synthesized a series of novel triple-modified 4-chloro-7-carbamatethiocolchicines. All the synthesized compounds have been tested in vitro to evaluate their cytotoxicity toward A549, MCF-7, LoVo, LoVo/DX, and BALB/3T3 cell lines. Additionally, their mode of binding to ß-tubulin was evaluated in silico. The majority of triple-modified colchicine derivatives exhibited significantly higher cytotoxicity than colchicine, doxorubicin, and cisplatin against tested cancerous cell lines with much higher selectivity index values for four of them.

Assessing and improving the performance of consensus docking strategies using the DockBox package.

Molecular docking is a well-established computational technique that aims to predict how a ligand binds to a specific protein target, as well as to assess the strength of the binding. Although docking programs are used worldwide for drug discovery, it is not always simple to identify which program or combination of programs provides the best results for a target of interest. Here we present DockBox, a computational package designed to facilitate the use of multiple docking and scoring programs allowing to combine them using different consensus strategies. As part of the DockBox package, a new consensus docking method called score-based consensus docking (SBCD) is introduced. SBCD was found to significantly improve the pose prediction success rates of single docking programs. When applied to virtual screening, SBCD enhanced enrichment factors while producing higher hit rates than standard consensus docking (CD). SBCD can be run with almost no additional computational cost and time compared to CD, if the same docking programs are used for pose generation. Furthermore, SBCD allows the use of many scoring functions to assess consensus without significant overhead, making it a promising new approach for the screening of large chemical libraries. DockBox is an open-source package publicly available at https://pypi.org/project/dockbox .

Synthesis, biological evaluation and molecular docking studies of new amides of 4-bromothiocolchicine as anticancer agents.

Colchicine is the major alkaloid isolated from the plant Colchicum autumnale, which shows strong therapeutic effects towards different types of cancer. However, due to the toxicity of colchicine towards normal cells its application is limited. To address this issue we synthesized a series of seven triple-modified 4-bromothiocolchicine analogues with amide moieties. These novel derivatives were active in the nanomolar range against several different cancer cell lines and primary acute lymphoblastic leukemia cells, specifically compounds: 5-9 against primary ALL-5 (IC50 = 5.3-14 nM), 5, 7-9 against A549 (IC50 = 10 nM), 5, 7-9 against MCF-7 (IC50 = 11 nM), 5-9 against LoVo (IC50 = 7-12 nM), and 5, 7-9 against LoVo/DX (IC50 = 48-87 nM). These IC50 values were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies revealed that colchicine and selected analogues induced characteristics of apoptotic cell death but manifested their effects in different phases of the cell cycle in MCF-7 versus ALL-5 cells. Specifically, while colchicine and the studied derivatives arrested MCF-7 cells in mitosis, very little mitotically arrested ALL-5 cells were observed, suggesting effects were manifest instead in interphase. We also developed an in silico model of the mode of binding of these compounds to their primary target, ß-tubulin. We conducted a correlation analysis (linear regression) between the calculated binding energies of colchicine derivatives and their anti-proliferative activity, and determined that the obtained correlation coefficients strongly depend on the type of cells used.

Probing the Basis of α-Synuclein Aggregation by Comparing Simulations to Single-Molecule Experiments.

Intrinsically disordered proteins often play an important role in protein aggregation. However, it is challenging to determine the structures and interactions that drive the early stages of aggregation because they are transient and obscured in a heterogeneous mixture of disordered states. Even computational methods are limited because the lack of ordered structure makes it difficult to ensure that the relevant conformations are sampled. We address these challenges by integrating atomistic simulations with high-resolution single-molecule measurements reported previously, using the measurements to help discern which parts of the disordered ensemble of structures in the simulations are most probable while using the simulations to identify residues and interactions that are important for oligomer stability. This approach was applied to α-synuclein, an intrinsically disordered protein that aggregates in the context of Parkinson's disease. We simulated single-molecule pulling experiments on dimers, the minimal oligomer, and compared them to force spectroscopy measurements. Force-extension curves were simulated starting from a set of 66 structures with substantial structured content selected from the ensemble of dimer structures generated at zero force via Monte Carlo simulations. The pattern of contour length changes as the structures unfolded through intermediate states was compared to the results from optical trapping measurements on the same dimer to discern likely structures occurring in the measurements. Simulated pulling curves were generally consistent with experimental data but with a larger number of transient intermediates. We identified an ensemble of ß-rich dimer structures consistent with the experimental data from which dimer interfaces could be deduced. These results suggest specific druggable targets in the structural motifs of α-synuclein that may help prevent the earliest steps of oligomerization.

Behavior of α, ß tubulin in DMSO-containing electrolytes.

α, ß-tubulin is a cytoskeletal protein that forms cylindrical structures termed microtubules, which are crucial to the cell for a variety of roles. Microtubules are frequently modelled as one-dimensional bionanowires that act as ion transporters in the cell. In this work, we used dynamic light scattering (DLS) to measure the hydrodynamic diameter of tubulin in the presence of a polar aprotic co-solvent. We found that the hydrodynamic diameter increased with increasing DMSO volume fraction, almost doubling at 20% DMSO. To evaluate if this was due to an enlarged solvation shell, we performed reference interaction site model (RISM) simulations and found that the extent of solvation was unchanged. Using fluorescence microscopy, we then showed that tubulin was polymerization competent in the presence of colchicine, and thus inferred the presence of oligomers in the presence of DMSO, which points to its mechanism of action as a microtubule polymerization enhancing agent. Tubulin oligomers are known to form when microtubules depolymerize and are controversially implicated in microtubule polymerization. We show that DLS may be used to monitor early-state microtubule polymerization and is a viable alternative to fluorescence and electron microscopy-based methods. Our findings showing that DMSO causes tubulin oligomerization are thus of critical importance, both for creating bio-inspired nanotechnology and determining its biophysical roles in the cell.

Synthesis and Biological Evaluation of Novel Triple-Modified Colchicine Derivatives as Potent Tubulin-Targeting Anticancer Agents.

Specific modifications of colchicine followed by synthesis of its analogues have been tested in vitro with the objective of lowering colchicine toxicity. Our previous studies have clearly shown the anticancer potential of double-modified colchicine derivatives in C-7 and C-10 positions. Here, a series of novel triple-modified colchicine derivatives is reported. They have been obtained following a four-step strategy. In vitro cytotoxicity of these compounds has been evaluated against four human tumor cell lines (A549, MCF-7, LoVo, and LoVo/DX). Additionally, the mode of binding of the synthesized compounds was evaluated in silico using molecular docking to a 3D structure of ß-tubulin based on crystallographic data from the Protein Data Bank and homology methodology. Binding free energy estimates, binding poses, and MlogP values of the compounds were obtained. All triple-modified colchicine derivatives were shown to be active at nanomolar concentrations against three of the investigated cancer cell lines (A549, MCF-7, LoVo). Four of them also showed higher potency against tumor cells over normal cells as confirmed by their high selectivity index values. A vast majority of the synthesized derivatives exhibited several times higher cytotoxicity than colchicine, doxorubicin, and cisplatin.

Tubulin's response to external electric fields by molecular dynamics simulations.

Tubulin heterodimers are the building blocks of microtubules and disruption of their dynamics is exploited in the treatment of cancer. Electric fields at certain frequencies and magnitudes are believed to do the same. Here, the tubulin dimer's response to external electric fields was determined by atomistic simulation. External fields from 50 to 750 kV/cm, applied for 10 ns, caused significant conformational rearrangements that were dependent upon the field's directionality. Charged and flexible regions, including the α:H1-B2 loop, ß:M-loop, and C-termini, were susceptible. Closer inspection of the α:H1-B2 loop in lower strength fields revealed that these effects were consistent and proportional to field strength, and the findings indicate that external electric fields modulate the stability of microtubules through conformational changes to key loops involved in lateral contacts. We also find evidence that tubulin's curvature and elongation are affected, and external electric fields may bias tubulin towards depolymerization.