Sulfobutylether-beta-cyclodextrin-enabled antiviral remdesivir: Characterization of electrospun- and lyophilized formulations.

Veklury™ by Gilead Sciences, Inc., containing antiviral drug, remdesivir (REM) has received emergency authorization in the USA and in Europe for COVID-19 therapy. Here, for the first time, we describe details of the non-covalent, host-guest type interaction between REM and the solubilizing excipient, sulfobutylether-beta-cyclodextrin (SBECD) that results in significant solubility enhancement. Complete amorphousness of the cyclodextrin-enabled REM formulation was demonstrated by X-ray diffraction, thermal analysis, Raman chemical mapping and electron microscopy/energy dispersive spectroscopy. The use of solubilizing carbohydrate resulted in a 300-fold improvement of the aqueous solubility of REM, and enhanced dissolution rate of the drug enabling the preparation of stable infusion solutions for therapy. 2D ROESY NMR spectroscopy provided information on the nature of REM-excipient interaction and indicated the presence of inclusion phenomenon and the electrostatic attraction between anionic SBECD and nitrogen-containing REM in aqueous solution.

Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation.

ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter.

Syntheses and antiproliferative effects of D-homo- and D-secoestrones.

Substituted and/or heterocyclic d-homoestrone derivatives were synthetized via the intramolecular cyclization of a δ-alkenyl-d-secoaldehyde, -d-secoalcohol or -d-secocarboxylic acid of estrone 3-benzyl ether. The d-secoalcohol was modified at three sites in the molecule. The in vitro antiproliferative activities of the new d-homo- and d-secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use of MTT assay. d-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against ovarian and cervical cell lines, respectively. Two d-secoestrones (6 and 12c) proved to be effective, with IC50 values comparable with those of the reference agent cisplatin. A selected compound (6) was tested by tubulin polymerization assay and its cancer specificity was additionally determined by using noncancerous human fibroblast cells.

Predicting substrates of the human breast cancer resistance protein using a support vector machine method.

BACKGROUND: Human breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) efflux transporter that confers multidrug resistance in cancers and also plays an important role in the absorption, distribution and elimination of drugs. Prediction as to if drugs or new molecular entities are BCRP substrates should afford a cost-effective means that can help evaluate the pharmacokinetic properties, efficacy, and safety of these drugs or drug candidates. At present, limited studies have been done to develop in silico prediction models for BCRP substrates. In this study, we developed support vector machine (SVM) models to predict wild-type BCRP substrates based on a total of 263 known BCRP substrates and non-substrates collected from literature. The final SVM model was integrated to a free web server. RESULTS: We showed that the final SVM model had an overall prediction accuracy of ~73% for an independent external validation data set of 40 compounds. The prediction accuracy for wild-type BCRP substrates was ~76%, which is higher than that for non-substrates. The free web server (http://bcrp.althotas.com) allows the users to predict whether a query compound is a wild-type BCRP substrate and calculate its physicochemical properties such as molecular weight, logP value, and polarizability. CONCLUSIONS: We have developed an SVM prediction model for wild-type BCRP substrates based on a relatively large number of known wild-type BCRP substrates and non-substrates. This model may prove valuable for screening substrates and non-substrates of BCRP, a clinically important ABC efflux drug transporter.

Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein.

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening.

Identification of a polyoxometalate inhibitor of the DNA binding activity of Sox2.

Aberrant expression of transcription factors is a frequent cause of disease, yet drugs that modulate transcription factor protein-DNA interactions are presently unavailable. To this end, the chemical tractability of the DNA binding domain of the stem cell inducer and oncogene Sox2 was explored in a high-throughput fluorescence anisotropy screen. The screening revealed a Dawson polyoxometalate (K(6)[P(2)Mo(18)O(62)]) as a direct and nanomolar inhibitor of the DNA binding activity of Sox2. The Dawson polyoxometalate (Dawson-POM) was found to be selective for Sox2 and related Sox-HMG family members when compared to unrelated paired and zinc finger DNA binding domains. [(15)N,(1)H]-Transverse relaxation optimized spectroscopy (TROSY) experiments coupled with docking studies suggest an interaction site of the POM on the Sox2 surface that enabled the rationalization of its inhibitory activity. The unconventional molecular scaffold of the Dawson-POM and its inhibitory mode provides strategies for the development of drugs that modulate transcription factors.

Drug transport by breast cancer resistance protein.

IMPORTANCE OF THE FIELD: The ATP-binding cassette transporter ABCG2 is a well-known major mediator of multi-drug resistance in cancers. Beyond multi-drug resistance, experimental and recent clinical studies demonstrate a role for ABCG2 as a determinant of drug pharmacokinetic, safety and efficacy profiles. AREAS COVERED IN THIS REVIEW: The clinical evidence of the role of ABCG2 in pharmacokinetics and pharmacodynamics is reviewed. Key questions that arise from the perspective of preclinical drug evaluation are addressed, including the structure of ABCG2 and mechanisms of drug-transporter interactions, mechanisms responsible for the polyspecificity of ABCG2, methods suitable for studying drug-ABCG2 interactions in vitro and in silico prediction of ABCG2 substrates and inhibitors. WHAT THE READER WILL GAIN: An update on current knowledge of the importance of ABCG2 in drug disposition with special emphasis on drug development. TAKE HOME MESSAGE: The field of drug-ABCG2 interaction is rapidly advancing and beginning to expand into clinical practice. However, the structural understanding of drug binding and transport by ABCG2 is still incomplete. Incorporation of novel concepts of drug-transporter interactions such as electrostatic funneling might help explain the multispecificity of ABCG2 and enable in silico predictions.

Functional adaptation of the switch-2 nucleotide sensor enables rapid processive translocation by myosin-5.

Active site loops that are conserved across superfamilies of myosins, kinesins, and G proteins play key roles in allosteric coupling of NTP hydrolysis to interaction with track filaments or effector proteins. In this study, we investigated how the class-specific natural variation in the switch-2 active site loop contributes to the motor function of the intracellular transporter myosin-5. We used single-molecule, rapid kinetic and spectroscopic experiments and semiempirical quantum chemical simulations to show that the class-specific switch-2 structure including a tyrosine (Y439) in myosin-5 enables rapid processive translocation along actin filaments by facilitating Mg(2+)-dependent ADP release. Using wild-type control and Y439 point mutant myosin-5 proteins, we demonstrate that the translocation speed precisely correlates with the kinetics of nucleotide exchange. Switch-2 variants can thus be used to fine-tune translocation speed while maintaining high processivity. The class-specific variation of switch-2 in various NTPase superfamilies indicates its general role in the kinetic tuning of Mg(2+)-dependent nucleotide exchange.

Homology modeling of breast cancer resistance protein (ABCG2).

BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.

Molecular modeling of the non-covalent binding of the dietary tomato carotenoids lycopene and lycophyll, and selected oxidative metabolites with 5-lipoxygenase.

Numerous studies on human prostate cancer cell lines indicate a role for arachidonic acid (AA) and its oxidative metabolites in prostate cancer proliferation. The metabolism of AA by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression, and metastasis. In particular, products of the 5-LOX pathway (including 5-HETE and 5-oxo-EET) have been implicated as potential 'survival factors' that may confer escape after androgen withdrawal therapy through fatty-acid (i.e., AA) drive. Potent natural dietary antioxidant compounds such as lycopene and lycophyll, with tissue tropism for human prostate, have been shown to be effective in ameliorating generalized oxidative stress at the DNA level. Suppressing the 5-LOX axis pharmacologically is also a promising avenue for intervention in human patients. The recently recognized direct interaction of the astaxanthin-based soft-drug Cardax to human 5-LOX with molecular modeling, and the downregulation of both 5-HETE and 5-oxo-EET in vivo in a murine peritonitis model, suggest that other important dietary carotenoids may share this enzyme regulatory feature. In the current study, the acyclic tomato carotene lycopene (in all-trans and 5-cis isomeric configurations) and its natural dihydroxy analog lycophyll (also present in tomato fruit) were subjected to molecular modeling calculations in order to investigate their predicted binding interaction(s) with human 5-LOX. Two bioactive oxidative metabolites of lycopene (4-methyl-8-oxo-2,4,6-nonatrienal and 2,7,11-trimethyl-tetradecahexaene-1,14-dial) were also investigated. A homology model of 5-LOX was constructed using 8-LOX and 15-LOX structures as templates. The model was validated by calculating the binding energy of Cardax to 5-LOX, which was demonstrated to be in good agreement with the published experimental data. Blind docking calculations were carried out in order to explore the possible binding sites of the carotenoids on 5-LOX, followed by focused docking to more accurately calculate the predicted energy of binding. Lycopene and lycophyll were predicted to bind with high affinity in the superficial cleft at the interface of the beta-barrel and the catalytic domain of 5-LOX (the 'cleavage site'). Carotenoid binding at this cleavage site provides the structural rationale by which polyenic compounds could modify the 5-LOX enzymatic function via an allosteric mechanism, or by radical scavenging in proximity to the active center. In addition, the two bioactive metabolites of lycopene were predicted to bind to the catalytic site with high affinity--therefore suggesting potential direct competitive inhibition of 5-LOX activity that should be shared by both lycopene and lycophyll after in vivo supplementation, particularly in the case of the dial metabolite.

Molecular modeling of non-covalent binding of homochiral (3S,3'S)-astaxanthin to matrix metalloproteinase-13 (MMP-13).

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of various important chronic illnesses, including cancer, arthritis, and cardiovascular disease (CVD). In particular, MMP-13 is currently being probed as a potential key target in CVD and malignant disease due to its documented effects on extracellular matrix (ECM) remodeling, important in the pathophysiology of these diseases. Within the family of related mammalian MMP enzymes, MMP-13 possesses a large hydrophobic binding pocket relative to that of other MMPs. Homochiral astaxanthin (3S,3'S-AST; 3S,3'S-dihydroxy-beta,beta-carotene-4,4'-dione), an important antioxidant and anti-inflammatory xanthophyll carotenoid, is an active metabolite of several novel soft drugs in clinical development; it is also extensively used and tested as a human nutraceutical. In the current study, the prediction of the geometry and energetics of its binding to human MMP-13 was conducted with molecular modeling. The method used was found to predict the energy of binding of known ligands of MMP-13 with great precision. Blind docking using the whole protein target was then used in order to identify the possible binding site(s) of AST. AST was predicted to bind at several sites in close proximity to the active center. Subsequent analyses focused on the binding site at the atomic (i.e., amino acid sequence) level suggested that AST can bind to MMP-13 with high affinity and favorable energetics. Therefore, the modeling study predicts potential direct enzyme-inhibitory activity of AST against MMP-13, a behavior that may be exploited in mammalian systems in which pathological upregulation of MMP activity is paramount.

Interactions of granisetron with an agonist-free 5-HT3A receptor model.

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.