Stability and in vitro antiproliferative activity of bioactive "Vitamin E" fortified parenteral lipid emulsions.

The objectives of this work were to engineer physically stable "Vitamin E" rich intravenous lipid emulsions and to evaluate their in vitro antiproliferative activity against MCF-7 (human mammary adenocarcinoma) and SW-620 (human colon adenocarcinoma) cell lines. Emulsions loaded with 70% vitamin E by total weight of the oil phase were stabilized with secondary emulsifiers and tested for their hemolytic effect and their plasma and electrolyte stability. Emulsions stabilized with sodium oleate and sodium deoxycholate were sensitive to electrolytes and exhibited significant hemolytic effect. On the other hand, addition of 2.5% poloxamer was found to stabilize the emulsions against electrolytes and physical stress, which was attributed to the steric effect of their polyoxyethylene (POE) head group. When tested for their antiproliferative effects, poloxamer-stabilized tocotrienol lipid emulsions were found to exhibit significantly higher anticancer activity than lipid emulsions enriched with tocopherol alone. The half maximal inhibitory concentrations (IC(50)) of tocotrienols lipid emulsions against MCF-7 and SW-620 were 14 and 12 µM, respectively, whereas the IC(50s) of tocopherol lipid emulsions were approximately 69 and 78 µM against MCF-7 and SW-620 cells, respectively.

Concurrent delivery of tocotrienols and simvastatin by lipid nanoemulsions potentiates their antitumor activity against human mammary adenocarcenoma cells.

Tocotrienol rich fraction (TRF) of vitamin E was previously shown to have anticancer activity against murine tumor cells in vitro. TRF was also shown to potentiate the anticancer activity of statins. The objectives of this study were therefore (a) to prepare and characterize stable parenteral lipid nanoemulsions as a novel platform for the concurrent delivery of TRF and simvastatin for subsequent use in combination chemotherapy, and (b) to evaluate the antiproliferative activity of the nanoemulsions against MCF-7 and MDA-MB-231 human mammary tumor cells. Nanoemulsions were prepared by the high-pressure homogenization technique using a viscous 70/30 blend of TRF and medium chain triglycerides as the oil phase in which simvastatin was dissolved at 9%w/w loading. Nanoemulsion droplets were about 200 nm in size and had surface potential of -45 mV. In a dissolution study, approximately 20% of simvastatin was released in sink conditions after 24h. The stability of the nanoemulsions was monitored over 6 months of storage. No oxidation or degradation products were detected and no loss in simvastatin loading was observed during this period. The antiproliferative activity of the nanoemulsions was also retained after storage. The IC50 of the TRF nanoemulsions against MCF-7 and MDA-MB-231 was 14 and 7 µM, respectively, which decreased to 10 µM and 4.8 µM when simvastatin was added to the nanoemulsions. Nanoemulsions prepared with tocopherol had no anticancer activity and were used as negative control. This study demonstrated that parenteral lipid nanoemulsions are viable delivery platform for potential use in cancer chemotherapy.

The opposite effects of doxorubicin on bone marrow stem cells versus breast cancer stem cells depend on glucosylceramide synthase.

Myelosuppression and drug resistance are common adverse effects in cancer patients with chemotherapy, and those severely limit the therapeutic efficacy and lead treatment failure. It is unclear by which cellular mechanism anticancer drugs suppress bone marrow, while drug-resistant tumors survive. We report that due to the difference of glucosylceramide synthase (GCS), catalyzing ceramide glycosylation, doxorubicin (Dox) eliminates bone marrow stem cells (BMSCs) and expands breast cancer stem cells (BCSCs). It was found that Dox decreased the numbers of BMSCs (ABCG2(+)) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24(-)/CD44(+)/ESA(+)) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1mg/kg/week, 42 days) decreased the numbers of BMSCs while it increased BCSCs in vivo. Breast cancer cells, rather than bone marrow cells, highly expressed GCS, which was induced by Dox and correlated with BCSC pluripotency. These results indicate that Dox may have opposite effects, suppressing BMSCs versus expanding BCSCs, and GCS is one determinant of the differentiated responsiveness of bone marrow and cancer cells.

Molecular modelling and multisimplex optimization of tocotrienol-rich self emulsified drug delivery systems.

The objective of this study was to optimize a novel tocotrienol (TRF)-rich self emulsified drug delivery system (SEDDS). In the first part, an unusual phenomenon was investigated. It was observed that by substituting Tween 80 with Cremophor EL in the SEDDS it was possible to emulsify>55% TRF (by weight of the formulation) into submicron (<200 nm) emulsion. With Tween, only 17.5% of the loaded TRF could be emulsified into crude emulsion. The superiority of Cremophor was attributed to the special arrangement of the surfactant at the oil/water interface, which was confirmed by modelling and docking studies. In the second part of this study, the composition of the secondary ingredients in the TRF-rich SEDDS were optimized by the modified Multisimplex approach. SEDDS were manufactured at pre-defined step-size and tested for their dissolution behavior. Testing was performed sequentially until the optimum composition that can emulsify 50% of the loaded TRF into a stable<150 nm submicron emulsion was obtained. Optimization end-point was identified when the "membership value" approached 1, which was confirmed by a second Multisimplex run. Overall, this study demonstrated the utility of docking studies and the Multisimplex approach in product development when little is known about the experimental "design space".

Medicinal chemistry for 2020.

Rapid advances in our collective understanding of biomolecular structure and, in concert, of biochemical systems, coupled with developments in computational methods, have massively impacted the field of medicinal chemistry over the past two decades, with even greater changes appearing on the horizon. In this perspective, we endeavor to profile some of the most prominent determinants of change and speculate as to further evolution that may consequently occur during the next decade. The five main angles to be addressed are: protein-protein interactions; peptides and peptidomimetics; molecular diversity and pharmacological space; molecular pharmacodynamics (significance, potential and challenges); and early-stage clinical efficacy and safety. We then consider, in light of these, the future of medicinal chemistry and the educational preparation that will be required for future medicinal chemists.

Structure-activity relationship of conformationally constrained peptidomimetics for antiproliferative activity in HER2-overexpressing breast cancer cell lines.

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases and is involved in a signaling cascade for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating the role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (compound 5, Arg-[3-amino-3(1-napthyl)-propionic acid]-Phe) exhibited antiproliferative activity with IC(50) values in the nanomolar to micromolar range in breast cancer cell lines. To further investigate the structure-activity relationship of the compounds, various analogs of compound 5 were designed. Conformational constraints were initiated in the peptidomimetic with introduction of a Pro residue in the peptidomimetic sequence. Results of antiproliferative activity indicated that analogs of compound 5 with C-and N-terminal ends capped (compound 16) and compound 9 with Asp at the C-terminal exhibited antiproliferative activity in the lower micromolar range against breast cancer cell lines. Introduction of conformational constraints such as Pro residue in the sequence or cyclization did not enhance the activity of the peptidomimetic. Competitive binding studies were carried out to evaluate the binding of potent peptidomimetics to HER2-overexpressing cancer cell lines. Results indicated that compounds exhibiting antiproliferative activity in breast cancer cell lines bind to the cells that overexpress HER2 protein.

Conformationally constrained peptides from CD2 to modulate protein-protein interactions between CD2 and CD58.

Cell adhesion molecule CD2 and its ligand CD58 provide good examples of protein-protein interactions in cells that participate in the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the ß-strand region of CD2 protein. The two strands were linked by a peptide bond. ß-Strands in the peptides were nucleated by inserting a ß-sheet-inducing (D)-Pro-Pro sequence or a dibenzofuran (DBF) turn mimetic with key amino acid sequences from CD2 protein that binds to CD58. The solution structures of the peptides (5-10) were studied by NMR and molecular dynamics simulations. The ability of these peptides to inhibit cell adhesion interaction was studied by E-rosetting and lymphocyte epithelial assays. Peptides 6 and 7 inhibit the cell adhesion activity with an IC(50) of 7 and 11 nM, respectively, in lymphocyte epithelial adhesion assay. NMR and molecular modeling results indicated that peptides 6 and 7 exhibited ß-hairpin structure in solution.

Heterotypic cell adhesion assay for the study of cell adhesion inhibition.

CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its ligand CD58 on antigen-presenting cells. Interaction between CD2 and CD58 or leukocyte function-associated antigen-3 (LFA-3) helps to optimize immune recognition facilitating contact between T lymphocytes and antigen-presenting cells. Modulation or inhibition of this interaction has been shown to be therapeutically useful in the treatment of autoimmune diseases. Antibodies and small molecules including peptides have been designed to modulate or disrupt the cell adhesion interactions due to CD2 and CD58. E-rosetting assay is a widely used method applied in the study of the modulation of CD2-CD58 interaction, which is either labor-intensive or radio-hazardous. In this chapter, we describe two methods that are used to study cell adhesion inhibition: (a) E-rosetting Assay and (b) Lymphocyte-epithelial assay. The second method, lymphocyte-epithelial assay, is a rapid and sensitive heterotypic cell adhesion assay for studying cell adhesion inhibition. The method relies on the CD2 expression on the surface of Jurkat cells and the CD58 expression on the surface of Caco-2 cells, which were confirmed by flow cytometry and ELISA studies respectively. This heterotypic cell adhesion assay described typically takes less than 4 h to perform, allows the evaluation of inhibitory activity of peptides/small molecules to modulate CD2-CD58 interaction in real cell system.

Active-learning exercises to teach drug-receptor interactions in a medicinal chemistry course.

OBJECTIVE: To incorporate structural biology, enzyme kinetics, and visualization of protein structures in a medicinal chemistry course to teach fundamental concepts of drug design and principles of drug action. DESIGN: Pedagogy for active learning was incorporated via hands-on experience with visualization software for drug-receptor interactions and concurrent laboratory sessions. Learning methods included use of clicker technology, in-class assignments, and analogies. ASSESSMENT: Quizzes and tests that included multiple-choice and open-ended items based on Bloom's taxonomy were used to assess learning. Student feedback, classroom exercises, and tests were used to assess teaching methods and effectiveness in meeting learning outcomes. CONCLUSION: The addition of active-learning activities increased students' understanding of fundamental medicinal chemistry concepts such as ionization state of molecules, enzyme kinetics, and the significance of protein structure in drug design.

A conformationally constrained peptidomimetic binds to the extracellular region of HER2 protein.

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases (other members include EGFR or HER1, HER3, and HER4) that are involved in signaling cascades for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating a role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (HERP5, Arg-beta Naph-Phe) exhibited antiproliferative activity with IC(50) values in the micromolar-to-nanomolar range in breast cancer cell lines. Binding of fluorescently labeled HERP5 to HER2 protein was evaluated by fluorescence assay, microscopy, and circular dichroism spectroscopy. Results indicated that HERP5 binds to the extracellular region of the HER2 protein. Structure of the peptidomimetic HERP5 was studied by NMR and molecular dynamics simulations. Based on these results a model was proposed for HER2-EGFR dimerization and possible blocking by HERP5 peptidomimetic using a protein-protein docking method.

A peptide from the beta-strand region of CD2 protein that inhibits cell adhesion and suppresses arthritis in a mouse model.

Cell adhesion molecules play a central role at every step of the immune response. The function of leukocytes can be regulated by modulating adhesion interactions between cell adhesion molecules to develop therapeutic agents against autoimmune diseases. Among the different cell adhesion molecules that participate in the immunologic response, CD2 and its ligand CD58 (LFA-3) are two of the best-characterized adhesion molecules mediating the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the beta-strand region of CD2 protein. The two strands were linked by a peptide bond. beta-Strands in the peptides were nucleated by inserting a beta-sheet-inducing Pro-Gly sequence with key amino acid sequences from CD2 protein that binds to CD58. Using a fluorescence assay, peptides that exhibited potential inhibitory activity in cell adhesion were evaluated for their ability to bind to CD58 protein. A model for peptide binding to CD58 protein was proposed based on docking studies. Administration of one of the peptides, P3 in collagen-induced arthritis in the mouse model, indicated that peptide P3 was able to suppress rheumatoid arthritis in mice.

Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

Design of beta-hairpin peptides for modulation of cell adhesion by beta-turn constraint.

The CD2-CD58 interaction in immune regulation and disease pathology has provided new targets for developing potential immunosuppressive agents. In the present study, we report the introduction of constraints to generate beta-hairpin structures from the strand sequences of CD2 protein. The beta-hairpin structures were induced in the designed peptides by introducing Pro-Gly sequences in the peptides. Results from NMR and MD simulation indicated that the peptides exhibited beta-turn structure at the X-Pro-Gly-Y sequence and formed the beta-hairpin structure in solution. The ability of these peptides to inhibit cell adhesion was evaluated by two cell adhesion assays. Among the peptides studied (1-4) (P1-P4), peptides 2-4 were able to inhibit cell adhesion between Jurkat cells and SRBC nearly 50% at 180 microM, and 80% inhibition between Jurkat cells and Caco-2 cells was seen at 90 microM. Peptide 1 did not show significant inhibition activity compared to control.

Novel peptide inhibitors of human secretory phospholipase A2 with antiinflammatory activity: solution structure and molecular modeling.

Secretory phospholipase A2 (sPLA2) and matrix metallopreoteinases (MMPs) are key enzymes involved in rheumatoid arthritis (RA), and their modulation thus represents a potential therapeutic option. On the basis of Escherichia coli radioassay, synthetic peptides were designed and screened for sPLA2 inhibition. The linear peptide, 10f (PIP-18), inhibited the recombinant human synovial sPLA2 activity with an IC50 of 1.19 microM. Not only did the peptide interfere with the function of sPLA2, but it also appeared to inhibit mRNA expression of sPLA2 and various MMPs in IL-1beta-stimulated RA synovial fibroblast (RASF) cultures and thereby the production of the corresponding proteins (>80% inhibition). Nuclear magnetic resonance (NMR), modeling, and docking studies indicate that in solution the peptide exhibits a beta-turn at residues Trp-Asp-Gly-Val and possibly binds to the hydrophobic channel of sPLA2. The results strongly suggest that the modulatory action of peptide 10f may play a major role in counteracting the development of RA.

Structure-function studies of peptides for cell adhesion inhibition: identification of key residues by alanine mutation and peptide-truncation approach.

Blockage of the interaction of CD2 with its ligand CD58 is expected to bring out potential therapeutic value for autoimmune diseases and organ transplantation. Three series of peptides (cVL, cIL and cAQ series) were designed from ratCD2 and humanCD2 to modulate CD2-CD58 interaction. To determine the specific segments in parent peptides responsible for inhibitory activity as lead sequence, we generated shorter fragments of the parent peptides and evaluated their biological activity with cell adhesion assay. The structure-activity relationship studies indicated that small cyclic peptides derived from CD2 ligand binding epitopes could mimic native beta-turn structure, and thus modulate CD2-CD58 interaction.

Structure-based rational design of beta-hairpin peptides from discontinuous epitopes of cluster of differentiation 2 (CD2) protein to modulate cell adhesion interaction.

Modulation or inhibition of interaction of cluster of differentiation (CD) adhesion molecules CD2-CD58 has been shown to be therapeutically useful. The analysis of the crystal structure of CD2 complexed with CD58 was carried out to define the epitopes that are important for the interaction of the two proteins. The crystal structure of CD2 indicated that the interaction surface of CD2 with CD58 has two beta-strand structures (F and C strands) with charged residues. On the basis of the crystal structure of the complex CD2-CD58, we have designed beta-hairpin peptides from the beta-strand region of CD2 by conjugating the discontinuous sequences in the protein. The peptides were modeled by molecular dynamics simulation, and their inhibitory activities were evaluated in vitro using two heterotypic cell adhesion assays, E-rosetting and lymphocyte-epithelial cell adhesion assays. Results indicated that 12- and 14-residue conjugate cyclic peptides cKS12 and cDD14 exhibited 60% and 50% inhibition activity, respectively, at 90 microM.

Structural analysis of peptides that interact with Newcastle disease virus.

A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide-virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive beta-turns around T2-L3-T4-T5 and K6-L7-Y8-C9. The other conformer exhibited a beta-hairpin bend type of structure with a bend around L3-T4-T5-K6.

Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: development of peptides for immunomodulation.

CD2 is a cell surface protein belonging to the immunoglobulin superfamily (IgSF) that plays a key role in mediating adhesion between human T-lymphocytes and target cells. The interaction between cell-adhesion molecules CD2 and CD58 is critical for immune response. Modulation or inhibition of these interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides and cyclic hexapeptides from the beta-turn and beta-strand region (hot spot) of human CD2 protein were designed to modulate CD2-CD58 interaction. The 12-amino acid synthetic cyclic peptides effectively blocked the interaction between CD2 and CD58 proteins as demonstrated by E-rosetting and heterotypic adhesion assays. NMR and molecular modeling studies indicated that these cyclic peptides exhibit beta-turn structure in solution and closely mimic the beta-turn structure of the surface epitopes of CD2 protein. The designed cyclic peptides with beta-turn structure have the ability to modulate CD2-CD58 interaction.