It remains extremely challenging to dissect the cooperative influence of multiple extracellular matrix (ECM) parameters on cell behavior. This stems in part from a lack of easily deployable strategies for the combinatorial variation of matrix biochemical and biophysical properties. Here we describe a simple, high-throughput platform based on light-modulated hyaluronic acid hydrogels that enables imposition of mutually independent and spatially continuous gradients of ligand density and substrate stiffness. We validate this system by showing that it can support mechanosensitive differentiation of mesenchymal stem cells. We then use it to show that the oncogenic microRNA, miR18a, is nonlinearly regulated by matrix stiffness and fibronectin density in glioma cells. The parallelization of this platform allows condensation of experiments that would normally require hundreds of independent hydrogels onto a single substrate. This system is a highly-accessible, high-throughput technique to study the combinatorial variation of biophysical and biochemical signals in a single experimental paradigm.
A highly efficient enantioselective C–H insertion of azavinyl carbenes into unactivated alkanes has been developed. These transition metal carbenes are directly generated from readily available and stable 1-sulfonyl-1,2,3-triazoles in the presence of chiral Rh(II) carboxylates and are used for C–H functionalization of alkanes to access a variety of β-chiral sulfonamides.
BackgroundCheckpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME).MethodsWe developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI.ResultsUsing a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs.ConclusionTaken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer.Statement of significanceCPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.
It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF 2 -substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF 2 -substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF 2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate∕Mg 2þ interaction over Watson-Crick hydrogen bonding was found and discussed.DNA polymerase beta | nonhydrolyzable nucleotides | fluorinated triphosphate | pentabasic acid | isopolarity and bioisotericity
Synthetically useful rhodium(II) carbenes were obtained from N-(1,2,4-triazolyl)-substituted 1,2,3-triazoles and Rh(II) carboxylates. The electron-withdrawing 1,2,4-triazolyl group reveals heretofore unknown reactivity of non-sulfonyl 1,2,3-triazoles, which exhibit reactivity of diazo compounds. The resulting carbenes provide ready asymmetric access to secondary homoaminocyclopropanes (80-95% ee, dr > 20:1) via reactions with olefins and also engage in efficient transannulation reactions with nitriles.
Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.
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