Understanding ligand binding, selectivity and functions on the G protein-coupled receptors: A molecular modeling approach

The assessment of target protein molecular structure provides a distinct advantage in the rational drug design process. The increasing number of available G protein-coupled receptor crystal structures has enabled utilization of a varied number of computational approaches for understanding the ligand-receptor interactions, ligand selectivity and even receptor response upon ligand binding. The following dissertation examines the results from three different projects with varied objectives – i) structural modeling of human C-C chemokine receptor type 5 (CCR5) and assessment of the ligand binding pocket of the receptor, ii) assessment of the selectivity profile of naltrexone derivatives on the three opioid receptors (μ-opioid, κ-opioid, δ-opioid) with an aim towards designing selective μ-opioid receptor antagonists, and iii) structural modeling of the ‘active’ state conformation of the κ-opioid receptor in response to agonist binding and determination of a plausible molecular mechanism involved in activation ‘switch’ of the κ-opioid receptor. In absence of a crystal-based molecular structure of CCR5, a homology model of the receptor was built and the ligand binding pocket was validated. On the basis of evaluation of the ligand-receptor interactions on the validated binding pocket, structural and chemical modifications to anibamine, a natural plant product, were proposed to enhance its receptor binding. The selectivity of naltrexone (a universal antagonist) was assessed with respect to the three opioid receptors by employing ligand docking studies and the ‘message-address’ concept. Multiple address sites were identified on the opioid receptors and structural modifications were proposed for the naltrexone derivatives for their enhanced selectivity. In the third project, structural modeling of the active state conformation of the κ-opioid receptor covalently bound to a salvinorin A derivative (agonist) was attempted via molecular dynamics simulations. Although the obtained molecular model lacked the signature ‘agonist-like’ conformations, the result provides a template for such studies in the future

Synthesis and Evaluation of Anibamine and Its Analogs as Novel Anti-Prostate Cancer Agents

The chemokine receptor CCR5 has been implicated in the pathogenesis of prostate cancer. A novel natural product, anibamine, was isolated and found to be a micromolar inhibitor of the receptor. Anibamine was used as a new anti-prostate cancer lead compound. To discover the pharmacophore, analogs of anibamine were designed using the “deconstruction-reconstruction-elaboration” approach and synthesized. The establishment of a stereoselective route to only one isomer was explored, to increase yield and eliminate elaborate purification procedures. Analogs were found to have anti-prostate cancer activity at levels higher than the parent compound. The molecular modeling studies of the deconstructed analogs indicate that due to the psuedo-symmetry of the parent compound, the binding conformation of the deconstructed analogs may not be very different from each other. All this information together may help identify a next generation lead compound for anti-prostate cancer treatment

DEVELOPMENT OF ANTAGONISTS TARGETING CHEMOKINE RECEPTOR CCR5 AND THE CHEMOKINE RECEPTOR CCR5 – MU OPIOID RECEPTOR HETERODIMER

The chemokine receptor CCR5 (CCR5) plays an integral role within the inflammatory network of cells. Importantly, CCR5 is a mediator in several disease states and can be targeted using small molecule antagonists. Within this work, CCR5’s role in prostate cancer and HIV/AIDS has been exploited in order to develop potential therapeutics and probes. First, a series of novel compounds was designed by using pharmacophore-based drug design based upon known CCR5 antagonists and molecular modeling studies of the CCR5 receptor’s three-dimensional conformation. Once synthesized, these compounds were tested for their CCR5 antagonism and their anti-proliferative effects in several prostate cancer cell lines. The data from both the calcium mobilization studies and the anti-proliferation studies suggests that the compounds synthesized have activity as CCR5 antagonists and as anti-proliferative agents in certain prostate cancer cell lines. In addition, a bivalent ligand containing both a mu opioid receptor (MOR) and a CCR5 antagonist pharmacophore was designed and synthesized in order to study the pharmacological profile of the putative CCR5-MOR heterodimer and its relation with NeuroAIDS. The structural-activity relationship between the bivalent ligand and the heterodimer was studied with radio-ligand binding assays, functional assays, HIV-1 fusion assays, cell fusion assays, and in silico molecular dynamics. The subsequent bivalent ligand was proven to be a potent inhibitor in both an artificial cell fusion assay mimicking HIV invasion and a native HIV-1 invasion assay using live virus. In all, two novel sets of compounds were synthesized that targeted either CCR5 or the CCR5-MOR heterodimer. For the CCR5 antagonists, as leads for prostate cancer therapeutics, further work needs to be done to ascertain and develop their structure-activity-relationship. This library of novel compounds was shown as promising leads as CCR5 and anti-prostate cancer agents. The bivalent ligand targeting the CCR5-MOR heterodimer proved to be a potent and tissue-specific inhibitor for neuroAIDS where the known treatment, maraviroc, is less efficacious and fails to inhibit virus entry in the presence of morphine. Both projects illustrate the roles that CCR5 plays in these two unique diseases

SYNTHESIS AND BIOLOGICAL EVALUATION OF SECOND GENERATION ANIBAMINE ANALOGUES AS NOVEL ANTI-PROSTATE CANCER AGENTS

Prostate cancer is the most prevalent non-cutaneous cancer among men. Since the 19th century when Virchow first introduced the concept of inflammation in cancer, chemokines and their receptors have garnered a lot of interest. Chemokine receptor CCR5 has been especially implicated in many disease states and recently found to be over expressed in prostate cancer cell lines. Anibamine, a natural CCR5 antagonist discovered in 2004, has been found to have significant anti-prostate cancer activity at micromolar level. To optimize this compound and also discover a novel pharmacophore, exploration of the original structure was carried out. Significant modifications were made to the side chain in the original structure and ten different analogues were prepared by altering the original synthetic route. While cytotoxicity assay proved the compounds to be non toxic to normal cells, anti-proliferation assay displayed that having a bulky, hydrophobic group in the side chain of the parent compound is essential for the activity. Looking at this data, the third generation of analogues can be prepared that might generate a better lead compound for the treatment of prostate cancer

Structural Insights from Binding Poses of CCR2 and CCR5 with Clinically Important Antagonists: A Combined In Silico Study

Chemokine receptors are G protein-coupled receptors that contain seven transmembrane domains. In particular, CCR2 and CCR5 and their ligands have been implicated in the pathophysiology of a number of diseases, including rheumatoid arthritis and multiple sclerosis. Based on their roles in disease, they have been attractive targets for the pharmaceutical industry, and furthermore, targeting both CCR2 and CCR5 can be a useful strategy. Owing to the importance of these receptors, information regarding the binding site is of prime importance. Structural studies have been hampered due to the lack of X-ray crystal structures, and templates with close homologs for comparative modeling. Most of the previous models were based on the bovine rhodopsin and β2-adrenergic receptor. In this study, based on a closer homolog with higher resolution (CXCR4, PDB code: 3ODU 2.5 Å), we constructed three-dimensional models. The main aim of this study was to provide relevant information on binding sites of these receptors. Molecular dynamics simulation was done to refine the homology models and PROCHECK results indicated that the models were reasonable. Here, binding poses were checked with some established inhibitors of high pharmaceutical importance against the modeled receptors. Analysis of interaction modes gave an integrated interpretation with detailed structural information. The binding poses confirmed that the acidic residues Glu291 (CCR2) and Glu283 (CCR5) are important, and we also found some additional residues. Comparisons of binding sites of CCR2/CCR5 were done sequentially and also by docking a potent dual antagonist. Our results can be a starting point for further structure-based drug design

Development of Bivalent Ligands Targeting the Putative CCR5-MOR Heterodimer

Chemokine receptor CCR5 (CCR5) is a G-protein coupled receptor (GPCR) predominantly expressed on leukocytes, or white blood cells.1–3 During inflammation, the body releases chemokines that bind to receptors such as CCR5 and attract leukocytes to the area of inflammation, leading to an immunological response.1 CCR5 is also an important receptor in the human immunodeficiency virus\u27s (HIV-1) invasion of host cells, as CCR5 acts as a co-receptor that facilitates HIV-1 viral entry.4,5 The continued destruction of leukocytes as a result of HIV-1 viral entry produces a disease state called acquired immunodeficiency syndrome (AIDS).5 Of note, this receptor is also expressed on the glial cells of the central nervous system (CNS).6,7 The mu-opioid receptor (MOR) is also a GPCR is predominantly expressed in the central nervous system.8–10 It binds to signal molecules such as endorphins and produces analgesic effects upon activation.9 The protein binds to morphine and morphine derivatives, which are extracts from the opium poppy plant.10 Besides the analgesic effects produced from MOR activation, morphine and its derivatives are also highly addictive and can result in drug dependence.11 Like CCR5, MOR is also expressed on the glial cells of the CNS.8 The accelerated progression of AIDS-like symptoms, in particular HIV-associated neurocognitive disorders (HANDS), has been observed in opiate-addicted patients.6,7,12–14 It has been discovered that opiate-addicted patients who have AIDS are susceptible to higher levels of HIV-1 viral proliferation and a greater level of CNS host cell destruction.12–14 This is because the activation of MOR by opiates appears to increase the expression of CCR5 on glial cells and may alter CCR5\u27s conformational state to one more susceptible to HIV-1 binding.15 Then, entry and subsequent destruction of glial cells by HIV-1 leads to the release of neurotoxic HIV-1 proteins that destroy primary neuronal cells.15 A bivalent ligand targeting the putative CCR5-MOR heterodimer was proposed to probe the interaction between the two proteins and act as a potential therapeutic ligand to combat neuroAIDS.16 A bivalent ligand attaching maraviroc, a CCR5 antagonist, with naltrexone, a non-selective opioid receptor antagonist, was synthesized and tested in vitro.16 The initial bivalent ligand was separated by a 21-atom spacer (Figure 1), the length of which was dictated by modeling studies and other bivalent ligands.17 The spacer was attached to the 6-position of 6β-naltrexamine, a modified variation of naltrexone replacing the 6 position ketone with an amino group, and the 4\u27-position of the maraviroc phenyl ring.16 These positions were chosen based on separate modeling studies of naltrexone and maraviroc docked in homology models of MOR and CCR5, respectively.18,19 From these studies, it appeared as if these positions were optimal given that they faced outward from their respective binding pockets and hence could tolerate spacer attachment.18,19 However, based on these modeling studies there was also room for structure optimization of the bivalent molecule.17–19 The original 21-atom spacer was subjected to numerous structural changes by our laboratory in an effort to increase CCR5 and MOR binding. The first type of structural modification included changing maraviroc\u27s point-of-attachment to the spacer from the 4\u27- to the 3\u27-position. Based on results from calcium mobilization functional assays involving CCR5-transfected human acute lymphoblastic leukemia (MOLT-4) cells, the activity of the bivalent molecule decreased from an IC50 of 126.0 ± 28.0 to 1340.0 ± 110.0 when the point-of-attachment was changed to the 3\u27-position. Thus, the 4\u27-position was kept in future structural studies. After this, additional structural modification was pursued in the form of changing spacer length. We synthesized two additional bivalent ligands, i.e., 19-atom and 23-atom bivalents with their controls. It is important to note that each of these molecules had a separate synthetic route starting with a specified diamine spacer. For the 19-atom bivalent molecule and its controls, the starting material was 1,5-diaminopentane. For the 23-atom bivalent molecule and its controls, the starting material was a 1,9-diaminononane molecule. Once these molecules were synthesized, in vitro biological testing was conducted. The bivalent molecules and 6β-naltrexamine controls were subjected to a competitive radioligand binding assay involving hMOR membranes and then to a calcium mobilization functional assay involving hMOR-transfected chinese hamster ovarian (CHO) cells (Figure 2). The affinities from the radioligand binding assay were similar in order-of-magnitude to other modified opioid antagonists and had a fold-decrease in affinity relative to naltrexone ranging from 1.1 to 9.3. The IC50 values from the calcium mobilization assay were similar in order-of-magnitude to other modified opioid antagonists and had a fold-decrease in activity relative to naltrexone ranging from 7.6 to 32. Thus, it was concluded that spacer attachment to the 6-position of 6β-naltrexamine was tolerated in MOR-binding. The bivalent molecules and maraviroc controls were then tested in calcium mobilization assays involving CCR5-transfed MOLT-4 cells to assess CCR5 activity. Unlike in the hMOR-CHO calcium mobilization assay, the activity of the bivalent molecules for CCR5 was not similar in magnitude to the receptor\u27s antagonist, maraviroc. The 23-atom bivalent and 19-atom bivalent had fold-decreases in activity relative to maraviroc of 1,100 and 250, respectively. Recently, the co-crystal structure of maraviroc bound to CCR5 was published in Science.20 Contrary to our previous understanding, it appeared as if modifications to the phenyl ring in maraviroc were not tolerated.20 After the biological testing, conformational analysis on the 19-, 21- and 23-atom bivalent compounds using Confort conformational modeling software was conducted. This was done to observe the possible viable conformations of each molecule. It was hypothesized that 1) the molecules could adopt viable conformations for binding to two different receptors simultaneously and that 2) the molecules could adopt similar conformations relative to each other. The first hypothesis was proposed to assess the realism of the project\u27s design strategy whereas the second was to analyze whether significant conformational differences could account for differences in binding activity between the three molecules. Results from this experiment showed that the molecules all adopted viable conformations for binding to two receptors simultaneously and that the conformational differences between the three molecules were negligible enough to conclude that significant differences in binding were not because of conformational differences. In conclusion, our laboratory synthesized a set of bivalent compounds to probe the CCR5-MOR heterodimer and tested such compounds in vitro. While spacer modifications to the 6-position of naltrexone were tolerated in hMOR competitive radioligand binding assays and in hMOR-CHO calcium mobilization assays, spacer modifications to maraviroc\u27s 4\u27-position on the phenyl ring were not tolerated very well in CCR5-MOLT-4 calcium mobilization assays. Therefore, future design strategies might focus on changing the spacer\u27s point-of-attachment to the maraviroc molecule

Sythesis and Biological Screening of a Series of Novel Chemokine Receptor CCR5 Antagonists

The chemokine receptor CCR5 has been implicated in the pathogenesis of cancers and AIDS. A series of novel piperidine derivatives were designed, synthesized, and evaluated as CCR5 antagonists. The ability of the new ligands to inhibit the increment of intracellular calcium level stimulated by endogenous ligand CCL5 was measured in the calcium mobilization assay as an indication of its CCR5 receptor antagonism. The anti-proliferation assay was performed to measure the ability of these new compounds to inhibit the proliferation of prostate cancer cell lines, PC-3 and M12. A new lead compound has been identified which showed micromolar level of inhibition to PC-3 cell line proliferation as well as calcium mobilization. These studies are the beginning of a thorough analysis of the CCR5 receptor antagonist binding pocket in the CCR5 receptor. Further examination may help identify next generation lead to develop highly selective CCR5 receptor antagonists and anti prostate cancer agents

Chemokine functionalized nanoparticles interactions with acute monocytic leukemia cells.

Biodegradable nanoparticles (NPs) aimed at drug/gene delivery can be functionalized with different molecules to have specific characteristics, like short bio-persistence, protection of the NP cargo and controlled release, specific cell/tissue targeting, as well as modulation of cellular signaling. To specifically improve NP selective targeting and modulation of immune responses, I focused my PhD project on the interactions between biocompatible NPs and immune cells. This goal was pursued by PLGA/Pluronic NP-surface decoration with two different chemokines, namely CXCL12 and CCL5. The first is a key mediator of homeostatic cell trafficking and it is involved in several pathologies, including cancers\u2019 metastasis. On the contrary, CCL5 is an induced chemokine attracting leukocytes during the inflammatory leukocyte effector phase. The rationale of the project relies on these molecules\u2019 roles to drive NPs towards specific cells exclusively expressing their cognate receptors, CXCR4 and CCR5. During the first two years I functionalized, characterized and tested the biological activity of chemokine-decorated PLGA-NPs in CXCR4 and CCR5 expressing human monocytic cells in vitro. I demonstrated CXCL12/CCL5-NPs stability in cell culture media and I proved that CXCL12-NPs enhanced binding/internalization abrogating CXCR4-mediated THP-1 leukemia cell chemotaxis. This result contributes to the search of biocompatible nanotools to quench tumor cell dysregulated migration. In the last year I optimized CCL5-NPs, further enhancing their cell targeting and internalization. I proved the dependence of CCL5-NP binding/internalization on the amount of surface-linked CCL5 and the unspecific adhesion of \u201cnaked\u201d PLGA/Pluronic-NPs

Trafficking regulation of the chemokine receptor CCR2B compared to CCR5

The closely-related CC chemokine receptors 2B and 5 are seven-transmembrane domain receptors coupled to heterotrimeric G proteins. The two receptors bind inflammatory chemokines and play important complementary roles in the recruitment of specific leukocyte sub-populations to sites of infection. To enable fine-tuning of cellular responses to chemokines, CCR2B and CCR5, like other GPCRs, can be desensitised in response to agonist stimulation or cross-talk with other receptors. This involves down-modulation of cell surface active receptor through two essential transportation events, endocytosis and recycling. The CCR5 endocytic and recycling pathways are well established and several mechanisms involved have been clearly defined. Conversely, less is known about the route followed by CCR2B upon stimulation. This study investigated the regulation, trafficking and fate of CCR2B in the context of THP-1 cells endogenously expressing the receptor and HEK293 transfectants. Comparison with CCR5 highlighted marked differences in the behavious of the two receptors. However, my initial findings indicate that certain aspects of the regulation of CCR5 as well as CCR2B may be cell type-dependent. Flow cytometry, immunofluorescence and biochemical analyses showed that unlike CCR5, internalised CCR2B can be both degraded and recycled following agonist stimulation. In HEK293, CCR2B follows an EGF receptor-like pathway, transiting through early endosomes containing EEA1, transferrin and Rab4, reaching CD63 and Lamp1 positive late endosomes/lysosomes before being degraded. Importantly, I showed that CCR2B cell surface molecules are N- and O-glycosylated, and only this glycosylated form of the receptor is targeted for agonist-induced degradation. This thesis also presents findings from proteomics approaches developed in an attempt to identify interacting proteins implicated in the trafficking of each receptor. This study brings new insights to the endocytic regulation of agonist-treated CC chemokine receptors, revealing receptor- and cell type-specific behaviours, which add complexity to a relatively conserved process

Immunotherapy of Folate Receptor Expressing Cancers

The folate receptor (FR) is a GPI anchored cell surface glycoprotein that functions to facilitate folic acid uptake and mediate signal transduction. With the introduction of multiple folate-targeted drugs into the clinic, the question has arisen regarding how frequently a patient can be dosed with a FR-targeted drug or antibody, and whether dosing frequency exerts any impact on the availability of FR for subsequent rounds of FR-mediated drug uptake. Although the rate of FR recycling has been examined in murine tumor models, little or no information exists on the impact of FR occupancy on its rate of endocytosis. The studies described in chapter two of this thesis quantitates the number of cell surface FR-α and FR-β following exposure to saturating concentrations of a variety of folate-linked molecules and anti-FR antibodies, including the unmodified vitamin, folate-linked drug mimetics, multi-folate derivatized nanoparticles, and monoclonal antibodies to FR. The collected data indicate that FR occupancy has no impact on the rate of FR internalization. The results also demonstrate that multivalent conjugates that bind and cross-link FRs at the cell surface internalize at the same rate as monovalent folate conjugates that have no impact on FR clustering, even though the multivalent conjugates traffic through a different endocytic pathway