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Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and natural killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a PET probe by radiolabeling a highly specific CD69 mAb, H1.2F3, with Zirconium-89 (89Zr), [89Zr]-deferoxamine (DFO)-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and IHC analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.

HIV/AIDS continues to pose an enormous burden on global health. Current HIV therapeutics include inhibitors that target the enzymes HIV protease, reverse transcriptase, and integrase, along with viral entry inhibitors that block the initial steps of HIV infection by preventing membrane fusion or virus-coreceptor interactions. With regard to the latter, peptides derived from the HIV coreceptor CCR5 were previously shown to modestly inhibit entry of CCR5-tropic HIV strains, with a peptide containing residues 178-191 of the second extracellular loop (peptide 2C) showing the strongest inhibition. Here we use an iterative approach of amino acid scanning at positions shown to be important for binding the HIV envelope, and recombining favorable substitutions to greatly improve the potency of 2C. The most potent candidate peptides gain neutralization breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than CCR5-tropic strains only. We found that gains in potency in the absence of toxicity were highly dependent on amino acid position and residue type. Using virion capture assays we show that 2C and the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies whose epitopes are located in or around variable loop 3 (V3) on gp120. Analysis of antibody binding data indicates that interactions between CCR5 ECL2-derived peptides and gp120 are localized around the base and stem of V3 more than the tip. In the absence of a high-resolution structure of gp120 bound to coreceptor CCR5, these findings may facilitate structural studies of CCR5 surrogates, design of peptidomimetics with increased potency, or use as functional probes for further study of HIV-1 gp120-coreceptor interactions.

Antibacterial-guided fractionation of the Dictyoceratid sponges Lamellodysidea sp. and two samples of Dysidea granulosa yielded 14 polybrominated, diphenyl ethers including one new methoxy-containing compound (8). Their structures were elucidated by interpretation of spectroscopic data of the natural product and their methoxy derivatives. Most of the compounds showed strong antimicrobial activity with low- to sub-microgram mL(-1) minimum inhibitory concentrations against drug-susceptible and drug-resistant strains of Staphylococcus aureus and Enterococcus faecium, and two compounds inhibited Escherichia coli in a structure-dependent manner.

Antibacterial-guided fractionation of an extract of a deep-water Topsentia sp. marine sponge led to the isolation of two new indole alkaloids, tulongicin A (1) and dihydrospongotine C (2), along with two known analogues, spongotine C (3) and dibromodeoxytopsentin (4). Their planar structures were determined by NMR spectroscopy. Their absolute configurations were determined through a combination of experimental and computational analyses. Tulongicin (1) is the first natural product to contain a di(6-Br-1H-indol-3-yl)methyl group linked to an imidazole core. The coexistence of tri-indole 1 and bis-indole alcohol 2 suggests a possible route to 1. All of the compounds showed strong antimicrobial activity against Staphylococcus aureus.

Carbohydrate binding proteins such as griffithsin, cyanovirin-N, and BanLec are potent HIV entry inhibitors and promising microbicides. Each binds to high-mannose glycans on the surface envelope glycoprotein gp120, yet the mechanisms by which they engage viral spikes and exhibit inhibition constants ranging from nanomolar to picomolar are not understood. To determine the structural and mechanistic basis for recognition and potency, we selected a panel of lectins possessing different valencies per subunit, oligomeric states, and relative orientations of carbohydrate binding sites to systematically probe their contributions to inhibiting viral entry. Cryo-electron micrographs and immuno gold staining of lectin-treated viral particles revealed two distinct effects—namely, viral aggregation or clustering of the HIV-1 envelope on the viral membrane—that were dictated by carbohydrate binding site geometry and valency. “Sandwich” surface plasmon resonance experiments revealed that a second binding event occurs only for those lectins that could aggregate viral particles. Furthermore, picomolar K(d) values were observed for the second binding event, providing a mechanism by which picomolar IC(50) values are achieved. We suggest that these binding and aggregation phenomena translate to neutralization potency.

In the absence of a cure or vaccine for HIV/AIDS, small molecule inhibitors remain an attractive choice for antiviral therapeutics. Recent structural and functional studies of the HIV-1 surface envelope glycoprotein gp120 have revealed sites of vulnerability that can be targeted by small molecule and peptide inhibitors, thereby inhibiting HIV-1 infection. Here we describe a series of small molecule entry inhibitors that were designed to mimic the sulfated N-terminal peptide of the HIV-1 coreceptor CCR5. From a panel of hydrazonothiazolyl pyrazolinones, we demonstrate that compounds containing naphthyl di- and tri-sulfonic acids inhibit HIV-1 infection in single round infectivity assays with the disulfonic acids being the most potent. Molecular docking supports the observed structure activity relationship, and SPR confirmed binding to gp120. In infectivity assays treatment with a representative naphthyl disulfonate and a disulfated CCR5 N-terminus peptide results in competitive inhibition, with combination indices >2. In total this work shows that gp120 and HIV-1 infection can be inhibited by small molecules that mimic the function of, and are competitive with the natural sulfated CCR5 N-terminus.