Recruitment of dynein and kinesin to viral particles.

Dynein and kinesin are cytoskeletal motor proteins involved in transporting cellular cargos and viruses. Throughout viral infection, they actively participate in the virus life cycle in the cell during entry, genome replication, and departure. Through their retrograde and anterograde transport, dynein and kinesin assist in promoting viral infection as well as the cellular defense response. This review highlights the crucial roles kinesin and dynein play in facilitating viral proliferation and aims to exhibit these proteins as vital targets for drug discovery in exploring strategies for regulating their dual functions concerning involvements in various essential phases of viral infections and host cells' immune response.

Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical "Toolkit".

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure-activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues' effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the "glutamate switch" mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

C-terminal Tail of ß-Tubulin and its Role in the Alterations of Dynein Binding Mode.

Dynein is a cytoskeletal molecular motor protein that moves along the microtubule (MT) and transports various cellular cargos during its movement. Using standard Molecular Dynamics (MD) simulation, Principle Component Analysis (PCA), and Normal Mode Analysis (NMA) methods, this investigation studied large-scale movements and local interactions of dynein's Microtubule Binding Domain (MTBD) when bound to tubulin heterodimer subunits. Examination of the interactions between the MTBD segments, and their adjustments in terms of intra- and intermolecular distances at the interfacial area with tubulin heterodimer, particularly at α-H16, ß-H18, and ß-tubulin C-terminal tail (CTT), was the main focus of this study. The specific intramolecular interactions, electrostatic forces, and the salt bridge residue pairs were shown to be the dominating factors in orchestrating movements of the MTBD and MT interfacial segments in the dynein's low-high-affinity binding modes. Important interactions included ß-Glu447 and ß-Glu449 (CTT) with Arg3469 (MTBD-H6), Lys3472 (MTBD-H6-H7 loop) and Lys3479 (MTBD-H7); ß-Glu449 with Lys3384 (MTBD-H8), Lys3386 and His3387 (MTBD-H1). The structural and precise position, orientation, and functional effects of the CTTs on the MT-MTBD, within reasonable cut-off distance for non-bonding interactions and under physiological conditions, are unavailable from previous studies. The absence of the residues in the highly flexible MT-CTTs in the experimentally solved structures is perhaps in some cases due to insufficient data from density maps, but these segments are crucial in protein binding. The presented work contributes to the information useful for the MT-MTBD structure refinement.

Metabolites of Vinca Alkaloid Vinblastine: Tubulin Binding and Activation of Nausea-Associated Receptors.

Vinblastine (VLB) is an antimitotic drug that binds to the vinca site of tubulin. The molecule possesses a high molecular weight and a complex chemical structure with many possibilities of metabolization. Despite advances in drug discovery research in reducing drug toxicity, the cause and mechanism of VLB-induced adverse drug reactions (ADRs) remains poorly understood. VLB is metabolized to at least 35 known metabolites, which have been identified and collected in this present work. This study also explores how VLB metabolites affect nausea-associated receptors such as muscarinic, dopaminergic, and histaminic. The metabolites have stronger binding interactions than acetylcholine (ACh) for muscarinic M1, M4, and M5 receptors and demonstrate similar binding profiles to that of the natural substrate, ACh. The affinities of VLB metabolites to dopaminergic and histaminic receptors, their absorption, distribution, metabolism, excretion, toxicity properties, and the superiority of VLB to ACh for binding to M5R, indicate their potential to trigger activation of nausea-associated receptors during chemotherapy with VLB. It has been shown that metabolite 20-hydroxy-VLB (metabolite 10) demonstrates a stronger binding affinity to the vinca site of tubulin than VLB; however, they have similar modes of action. VLB and metabolite 10 have similar gastric solubility (FaSSGF), intestinal solubility (FeSSIF), and log P values. Metabolite 10 has a more acceptable pharmacokinetic profile than VLB, a better gastric and intestinal solubility. Furthermore, metabolite 10 was found to be less bound to plasma proteins than VLB. These are desired and essential features for effective drug bioavailability. Metabolite 10 is not a substrate of CYP2D6 and thus is less likely to cause drug-drug interactions and ADRs compared to its parent drug. The hydroxyl group added upon metabolism of VLB suggests that it can also be a reasonable starting compound for designing the next generation of antimitotic drugs to overcome P-glycoprotein-mediated multidrug resistance, which is often observed with vinca alkaloids.

Interdomain twists of human thymidine phosphorylase and its active-inactive conformations: Binding of 5-FU and its analogues to human thymidine phosphorylase versus dihydropyrimidine dehydrogenase.

5-fluorouracil (5-FU) is an anticancer drug, which inhibits human thymidine phosphorylase (hTP) and plays a key role in maintaining the process of DNA replication and repair. It is involved in regulating pyrimidine nucleotide production, by which it inhibits the mechanism of cell proliferation and cancerous tumor growth. However, up to 80% of the administered drug is metabolized by dihydropyrimidine dehydrogenase (DPD). This work compares binding of 5-FU and its analogues to hTP and DPD, and suggests strategies to reduce drug binding to DPD to decrease the required dose of 5-FU. An important feature between the proteins studied here was the difference of charge distribution in their binding sites, which can be exploited for designing drugs to selectively bind to the hTP. The 5-FU presence was thought to be required for a closed conformation. Comparison of the calculation results pertaining to unliganded and liganded protein showed that hTP could still undergo open-closed conformations in the absence of the ligand; however, the presence of a positively charged ligand better stabilizes the closed conformation and rigidifies the core region of the protein more than unliganded or neutral liganded system. The study has also shown that one of the three hinge segments linking the two major α and α/ß domains of the hTP is an important contributing factor to the enzyme's open-close conformational twist during its inactivation-activation process. In addition, the angle between the α/ß-domain and the α-domain has shown to undergo wide rotations over the course of MD simulation in the absence of a phosphate, suggesting that it contributes to the stabilization of the closed conformation of the hTP.

Drug metabolites and their effects on the development of adverse reactions: Revisiting Lipinski's Rule of Five.

Many studies have shown that toxicities of anticancer drugs and their adverse effects are related to their chemical structure and high molecular weight that may result in a number of metabolites interacting with drug off-target networks. These factors require further attention for advancing cancer treatment and decreasing toxicities caused by the molecular complexity of antineoplastic agents. Providing more target-selective and tolerable cancer therapy with fewer side effects would not only improve patients' compliance, but also would decrease cancer-remission rates. This review presents several antineoplastic agents and their metabolites with molecular weights greater than 500 g/mol, which reportedly cause more than fifteen types of adverse reactions during breast cancer therapy.

Mechanism of Off-Target Interactions and Toxicity of Tamoxifen and Its Metabolites.

Tamoxifen is an estrogen modulator that acts to competitively inhibit the binding of endogenous estrogens. It is widely used for treatment of breast cancer; however, analogous with many antineoplastic agents, tamoxifen is associated with numerous adverse effects, most prominently nausea. We have identified several off-target receptors of tamoxifen and 22 of its metabolites that include histamine H1 and H3, and muscarinic M1, M4, and M5 subtypes, and dopamine D2 receptor. We have shown how they are associated with tamoxifen and its metabolites' toxicity through a comprehensive computational analysis of their interaction modes, which were also compared to that of the related endogenous substrates of each receptor. The results were further evaluated using available in vivo and in vitro data. The presented work provides foundational knowledge toward the determination of the precise mechanism of nausea induction, and in particular, interactions of tamoxifen and its metabolites with the receptors involved in that biomolecular pathway. This study can assist in predicting the potential undesired effects of the chemicals with common pharmacophores or similar fragments to that of tamoxifen and its metabolites and serve drug discovery research in developing more effective and tolerable tamoxifen analogues or chemotherapeutic agents.

Functionalized single-walled carbon nanotubes: cellular uptake, biodistribution and applications in drug delivery.

The unique properties of single-walled carbon nanotubes (SWNTs) enable them to play important roles in many fields. One of their functional roles is to transport cargo into cell. SWNTs are able to traverse amphipathic cell membranes due to their large surface area, flexible interactions with cargo, customizable dimensions, and surface chemistry. The cargoes delivered by SWNTs include peptides, proteins, nucleic acids, as well as drug molecules for therapeutic purpose. The drug delivery functions of SWNTs have been explored over the past decade. Many breakthrough studies have shown the high specificity and potency of functionalized SWNT-based drug delivery systems for the treatment of cancers and other diseases. In this review, we discuss different aspects of drug delivery by functionalized SWNT carriers, diving into the cellular uptake mechanisms, biodistribution of the delivery system, and safety concerns on degradation of the carriers. We emphasize the delivery of several common drugs to highlight the recent achievements of SWNT-based drug delivery.

Insight into the mechanism of chemical modification of antibacterial agents by antibiotic resistance enzyme O-phosphotransferase-IIIA.

In the present work, the mechanism of resistance to aminoglycoside antibiotics was investigated. We examined the conformational changes of the O-phosphotransferase-IIIa enzyme, complexed with the antibiotics using MD simulations. The inhibitory effects of a group of antibacterial peptides against the enzyme were also examined, among which CP10A showed the highest affinity and the results correlated with the measured IC50 values. The regioselectivity of the phosphorylation reaction was shown to be in favor of the OH at the 5″ position versus the 3' of the antibiotic. The binding mode of CP10A was evaluated by means of MD simulation that resulted in recognizing its Trp8 and Arg13 residues binding near to where residues at the 3' and 5″ positions of the antibiotic would bind; thus, they are essential for the peptide inhibitory effect. The major open, semi-open, and closed conformations of the binding sites were identified throughout the MD trajectory, which enable the enzyme to regulate the influx of molecules into these sites. Based on the enzyme crystal structure, it was assumed that the 'antibiotic loop' of the enzyme is stable in its liganded mode; however, MD results revealed that the loop is highly flexible in both liganded and ligand-free modes.

Molecular assembly of lethal factor enzyme and pre-pore heptameric protective antigen in early stage of translocation.

During intoxication, the anthrax toxin lethal (LF) and edema (EF) factors initially assemble with the protective antigen (PA) on the plasma membrane of cells expressing the membrane-bound surface-exposed anthrax toxin receptor (ATR). This takes place at the physiological pH prior to entering the acidic environment of the endosome. We elucidated the molecular dynamics (MD) behaviors of the three-dimensional structure of the (PA63)7LF3 complex in various conformations and analyzed the dynamical properties of the fully loaded pre-pore complex on the plasma membrane at the physiological pH. The analysis points to the interaction networks of amino acids conserved between PA63 octamer and heptamer, which are not affected during the initial stage of the LFs binding. The simulations show an asymmetrical movement of the complex domains that directly affect LFs conformations. The conformational and structural alterations of the 2ß2-2ß3 loops of PA subunits are associated with pore formation. The early conformational changes of the loops appear as they peel off from the domain 2 toward domain 4 of each PA subunit. The LFs unfold in 1α1 segments of their N-terminal initiating the early stage of the pre-pore formation. The results indicate instable regions within the complex and provide important clues concerning the detail of fluctuating residues of the LF-PA interface regions at the early steps of toxins translocation.

Dynamic change of heme environment in soluble guanylate cyclase and complexation of NO-independent drug agents with H-NOX domain.

Soluble guanylate cyclase is a heterodimer receptor that functions in several signal transduction pathways. Conversion of guanosine 5'-triphosphate to 3',5'-cyclic monophosphate second messenger at the catalytic domain is regulated by the changes at heme nitric oxide/oxygen domain of the ß-subunit. To better understand conformational changes at heme site that may impact on activities of catalytic domain, three soluble guanylate cyclase homolog proteins with heme at Fe-His state were investigated, and their dynamic behaviors were monitored in both unliganded (apo) and complex with heme. As a result of dynamic conformational changes, Lys110, Asp45, Arg135, and Glu41 were found interacting with the site gate, which may interfere with transportation of small molecules in and out of the heme site. An alternative binding site adjacent to that of heme was identified. Binding affinity of several nitric oxide-independent activators and heme-dependent stimulators was examined, and their binding modes in the heme site and in the alternative binding site in the human soluble guanylate cyclase enzyme were computationally simulated. The calculated binding energies were used as criteria to filter results of virtual high-throughput screenings based on FlexX ligand-docking algorithm and absorption, distribution, metabolism, excretion, and toxicity properties on databases of available drugs. The identified drugs from virtual high-throughput screening have been suggested for experimental investigations, based on which they may either be directly repurposed or require structural modifications for better physico-chemical and pharmacological properties.

Modeling the yew tree tubulin and a comparison of its interaction with paclitaxel to human tubulin.

PURPOSE: To explore possible ways in which yew tree tubulin is naturally resistant to paclitaxel. While the yew produces a potent cytotoxin, paclitaxel, it is immune to paclitaxel's cytotoxic action. METHODS: Tubulin sequence data for plant species were obtained from Alberta 1000 Plants Initiative. Sequences were assembled with Trinity de novo assembly program and tubulin identified. Homology modeling using MODELLER software was done to generate structures for yew tubulin. Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann calculations were performed with the Amber package to determine binding affinity of paclitaxel to yew tubulin. ClustalW2 program and PHYLIP package were used to perform phylogenetic analysis on plant tubulin sequences. RESULTS: We specifically analyzed several important regions in tubulin structure: the high-affinity paclitaxel binding site, as well as the intermediate binding site and microtubule nanopores. Our analysis indicates that the high-affinity binding site contains several substitutions compared to human tubulin, all of which reduce the binding energy of paclitaxel. CONCLUSIONS: The yew has achieved a significant reduction of paclitaxel's affinity for its tubulin by utilizing several specific residue changes in the binding pocket for paclitaxel.

Peloruside, laulimalide, and noscapine interactions with beta-tubulin.

This article reviews the recent findings regarding the binding sites, binding modes and binding affinities of three novel antimitotic drugs peloruside, laulimalide and noscapine with respect to tubulin as the target of their action. These natural compounds are shown to bind to ß-tubulin and stabilize microtubules for the cases of peloruside A and laulimalide, and prolong the time spent in pause for noscapine. Particular attention is focused on ß-tubulin isotypes as targets for new cancer chemotherapy agents and the amino acid differences in the binding site for these compounds between isotypes. We propose a new strategy for antimitotic drug design that exploits differential distributions of tubulin isotypes between normal and cancer cells and corresponding differential affinities between various drug molecules and tubulin isotypes.

Full-length structural model of RET3 and SEC21 in COPI: identification of binding sites on the appendage for accessory protein recruitment motifs.

COPI, a 600 kD heptameric complex (consisting of subunits α, ß, γ, δ, ε, ζ, and ß') "coatomer," assembles non-clathrin-coated vesicles and is responsible for intra-Golgi and Golgi-to-ER protein trafficking. Here, we report the three-dimensional structures of the entire sequences of yeast Sec21 (γ-COPI mammalian ortholog), yeast Ret3 (ζ-COPI mammalian ortholog), and the results of successive molecular dynamics investigations of the subunits and assembly based on a protein-protein docking experiment. The three-dimensional structures of the subunits in their complexes indicate the residues of the two subunits that impact on assembly, the conformations of Ret3 and Sec21, and their binding orientations in the complexed state. The structure of the appendage domain of Sec21, with its two subdomains--the platform and the ß-sandwich, was investigated to explore its capacity to bind to accessory protein recruitment motifs. Our study shows that a binding site on the platform is capable of binding the Eps15 DPF and epsin DPW2 peptides, whereas the second site on the platform and the site on the ß-sandwich subdomain were found to selectively bind to the amphiphysin FXDXF and epsin DPW1 peptides, respectively. Identifying the regions of both the platform and sandwich subdomains involved in binding each peptide motif clarifies the mechanism through which the appendage domain of Sec21 engages with the accessory proteins during the trafficking process of non-clathrin-coated vesicles.

Determination of noscapine's localization and interaction with the tubulin-α/ß heterodimer.

Noscapine, the benzylisoquinoline alkaloid, 5-(4,5-Dimethoxy-3-oxo-1,3-dihydro-isobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-6-ium, has been extensively used as a cough-suppressing medication with low toxicity. It has been recently shown to also have anti-cancer activity in mice and humans. In this work, using in silico analyses, the most probable binding site for noscapine is identified to be at the intradimer region of the α and ß subunits of the tubulin heterodimer. By utilization of small molecule docking techniques, and an analysis of the thermodynamically favorable binding modes of noscapine in its binding site, the key residues of tubulin monomers interacting with noscapine are determined. Upon noscapine binding, the conformational change in the tubulin heterodimer along with a potential long-range allosteric effect on both the N and E sites is studied by means of molecular dynamics simulations. Noscapine is found to function as a tubulin-stabilizing agent that interacts strongest with the lateral and longitudinal segments of the tubulin dimer, impacting the interaction between monomers in neighboring protofilaments. We infer that this may act as a depolymerization inhibitor of microtubules. As a result of this study, we have designed novel analogues of noscapine with the ultimate goal of finding agents with increased anti-tumor activity and lower inhibitory concentrations than that of noscapine.

Protein tyrosine phosphatases are regulated by mononuclear iron dicitrate.

The involvement of macrophages (Mvarphis) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mvarphi protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)(2)H(4-x)]((1+x)-) as the species responsible for the specific inhibition of Mvarphi PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)(2)](5-) targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.

A QXP-based multistep docking procedure for accurate prediction of protein-ligand complexes.

The two great challenges of the docking process are the prediction of ligand poses in a protein binding site and the scoring of the docked poses. Ligands that are composed of extended chains in their molecular structure display the most difficulties, predominantly because of the torsional flexibility. On the basis of the molecular docking program QXP-Flo+0802, we have developed a procedure particularly for ligands with a high degree of rotational freedom that allows the accurate prediction of the orientation and conformation of ligands in protein binding sites. Starting from an initial full Monte Carlo docking experiment, this was achieved by performing a series of successive multistep docking runs using a local Monte Carlo search with a restricted rotational angle, by which the conformational search space is limited. The method was established by using a highly flexible acetylcholinesterase inhibitor and has been applied to a number of challenging protein-ligand complexes known from the literature.

Molecular docking study on the "back door" hypothesis for product clearance in acetylcholinesterase.

Acetylcholinesterase (AChE) is one of the fastest enzymes known, even though the active site is buried inside the protein at the end of a 20-A deep narrow gorge. Among the great variety of crystal structures of this enzyme, both in the absence and presence of various ligands and proteins, the structure of a complex of AChE with the pseudo-irreversible inhibitor Mf268 is of particular interest, as it assists in the proposal of a back door for product clearance from the active site. Binding of Mf268 to AChE results in the carbamoylation of Ser200 and liberation of an eseroline-fragment as the leaving group. The crystal structure of the AChE-Mf268 complex, however, proves that eseroline has escaped from the enzyme, despite the fact that the Ser-bound inhibitor fragment blocks the gorge entrance. The existence of alternative routes other than through the gorge for product clearance has been postulated but is still controversially discussed in the literature, as an experimental proof for such a back door is still missing. We have used Monte Carlo-based molecular docking methods in order to examine possible alternative pathways that could allow eseroline to be released from the protein after being cleaved from the substrate by Ser200. Based on our results, a short channel at the bottom of the gorge seems to be the most probable back-door site, which begins at amino acid Trp84 and ends at the enzyme surface in a cavity close to amino acid Glu445. [Figure: see text].