The cannabinoid receptor 2 (CB2) plays an important role in the immune system. Although a few of GPCRs crystallographic structures have been reported, it is still challenging to obtain functional transmembrane proteins and high resolution X-ray crystal structures, such as for the CB2 receptor. In the present work, we used 10 reported crystal structures of GPCRs which had high sequence identities with CB2 to construct homology-based comparative CB2 models. We applied these 10 models to perform a prescreen by using a training set consisting of 20 CB2 active compounds and 980 compounds randomly selected from the National Cancer Institute (NCI) database. We then utilized the known 170 cannabinoid receptor 1 (CB1) or CB2 selective compounds for further validation. Based on the docking results, we selected one CB2 model (constructed by β1AR) that was most consistent with the known experimental data, revealing that the defined binding pocket in our CB2 model was well-correlated with the training and testing data studies. Importantly, we identified a potential allosteric binding pocket adjacent to the orthosteric ligand-binding site, which is similar to the reported allosteric pocket for sodium ion Na+ in the A2AAR and the δ-opioid receptor. Our studies in correlation of our data with others suggested that sodium may reduce the binding affinities of endogenous agonists or its analogs to CB2. We performed a series of docking studies to compare the important residues in the binding pockets of CB2 with CB1, including antagonist, agonist, and our CB2 neutral compound (neutral antagonist) XIE35-1001. Then, we carried out 50 ns molecular dynamics (MD) simulations for the CB2 docked with SR144528 and CP55940, respectively. We found that the conformational changes of CB2 upon antagonist/agonist binding were congruent with recent reports of those for other GPCRs. Based on these results, we further examined one known residue, Val1133.32, and predicted two new residues, Phe183 in ECL2 and Phe2817.35, that were important for SR144528 and CP55940 binding to CB2. We then performed site-directed mutation experimental study for these residues and validated the predictions by radiometric binding affinity assay.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist that shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nanoscale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer, poly­[di­(carboxylato­phenoxy)­phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic light scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and 1H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response toward desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The noncovalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.