Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death.

Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands.

Multivalent Fluorinated Nanorings for On-Cell F NMR.

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in F magnetic resonance imaging (MRI)-based molecular imaging. Chemically self-assembled nanorings (CSANs) provide a strategy to increase the fluorine content through multivalent display. We previously reported an F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E-dimeric dihydrofolate (E-DD) was bioconjugated to a highly fluorinated peptide.

The Many Faces of Histidine Triad Nucleotide Binding Protein 1 (HINT1).

The histidine triad nucleotide binding protein 1 (HINT1) is a nucleoside phosphoramidase that has garnered interest due to its widespread expression and participation in a broad range of biological processes. Herein, we discuss the role of HINT1 as a regulator of several CNS functions, tumor suppressor, and mast cell activator via its interactions with multiple G-protein-coupled receptors and transcription factors. Importantly, altered HINT1 expression and mutation are connected to the progression of multiple disease states, including several neuropsychiatric disorders, peripheral neuropathy, and tumorigenesis.

Design of Highly Fluorinated Peptides for Cell-based F NMR.

The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength.

The eIF4EBP-eIF4E axis regulates CD4 T cell differentiation through modulation of T cell activation and metabolism.

CD4 T cells are critical for adaptive immunity, differentiating into distinct effector and regulatory subsets. Although the transcriptional programs underlying their differentiation are known, recent research has highlighted the importance of mRNA translation in determining protein abundance. We previously conducted genome-wide analysis of translation in CD4 T cells revealing distinct translational signatures distinguishing these subsets, identifying eIF4E as a central differentially translated transcript.

Eradication of Heterogeneous Tumors by T Cells Targeted with Combination Bispecific Chemically Self-assembled Nanorings.

Cancer stem-like cells (CSCs) are often the root cause of refractive relapse due to their inherent resistance to most therapies and ability to rapidly self-propagate. Recently, the antigen CD133 has been identified as a CSC marker on several cancer types and αCD133 therapies have shown selective targeting against CSCs with minimal off-target toxicity. Theoretically, by selectively eliminating CSCs, the sensitivity to bulk tumor-targeting therapies should be enhanced.

Engineering Biomimetic Trogocytosis with Farnesylated Chemically Self-Assembled Nanorings.

Inspired by the natural intercellular material-transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (f-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells, thus allowing cell-cell interactions to be monitored and facilitating cell-cell communications. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted f-CSANs, which were efficiently transferred to receiver cells expressing the appropriate receptors by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor specific and dependent on direct cell-cell interactions, the rate of receptor internalization, and the level of receptor expression.

Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides.

Human histidine triad nucleotide-binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100), which gate a conserved water channel leading to the active site 13 Å away.

Manipulating Cell Fates with Protein Conjugates.

The homeostasis of cellular activities is essential for the normal functioning of living organisms. Hence, the ability to regulate the fates of cells is of great significance for both fundamental chemical biology studies and therapeutic development. Despite the notable success of small-molecule drugs that normally act on cellular protein functions, current clinical challenges have highlighted the use of macromolecules to tune cell function for improved therapeutic outcomes.

Multivalent, Bispecific αB7-H3-αCD3 Chemically Self-Assembled Nanorings Direct Potent T Cell Responses against Medulloblastoma.

Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3.

Engineering reversible cell-cell interactions using enzymatically lipidated chemically self-assembled nanorings.

Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions.

"Switching On" Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor.

Enzymatically driven change to the spectroscopic properties of a chemical substrate or product has been a linchpin in the development of continuous enzyme kinetics assays. These assays inherently necessitate substrates or products that naturally comply with the constraints of the spectroscopic technique being used, or they require structural changes to the molecules involved to make them observable. Here we demonstrate a new analytical kinetics approach with enzyme histidine triad nucleotide binding protein 1 (HINT1) that allows us to extract both useful values and a rank-ordered list of substrate specificities without the need to track substrates or products directly.

4Ei-10 interdiction of oncogenic cap-mediated translation as therapy for non-small cell lung cancer.

In order to suppress 5' cap-mediated translation a highly available inhibitor of the interaction between the 5' mRNA cap and the eIF4E complex has been developed. 4Ei-10 is a member of the class of ProTide compounds and has elevated membrane permeability and is a strong active chemical antagonist for eIF4E. Once taken up by cells it is converted by anchimeric activation of the lipophilic 2-(methylthio) ethyl protecting group and after that Hint1 P-N bond cleavage to N-(p-chlorophenoxyethyl) guanosine 5'-monophosphate (7-Cl-Ph-Ethyl-GMP).

A Phosphoramidate Strategy Enables Membrane Permeability of a Non-nucleotide Inhibitor of the Prolyl Isomerase Pin1.

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides.

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T Cell-Mediated Antitumor Responses and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression.

Overexpression of the epidermal growth factor receptor (EGFR) on various cancers makes it an important target for cancer immunotherapy. We recently demonstrated that single-chain variable fragment-based bispecific chemically self-assembled nanorings (CSANs) can successfully modify T cell surfaces and function as prosthetic antigen receptors (PARs) allowing selective targeting of tumor antigens while incorporating a dissociation mechanism of the rings. Here, we report the generation of anti-EGFR fibronectin (FN3)-based PARs with high yield, rapid protein production, predicted low immunogenicity, and increased protein stability.

and Characterization of the Anti-Zika Virus Activity of ProTides of 2'-C-β-Methylguanosine.

The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-β-methylguanosine. ProTide is a 2'-C-β-methylguanosine tryptamine phosphoramidate monoester, and ProTide is a 2-(methylthio)-ethyl-2'-C-β-methylguanosine tryptamine phosphoramidate diester.

Engineering reversible cell-cell interactions with chemical biology.

Techniques to direct cell-cell interactions have advanced our understanding of fundamental biology and opened new avenues in tissue engineering, regenerative medicine, and immunotherapy. This is often achieved by introducing new targeting ligands to the cell membrane, which can be accomplished through both genetic and nongenetic approaches. While both offer advantages, nongenetic modifications tend to be faster to produce, innocuous to the modified cell, and potentially reversible.

Profiling of Small Ribosomal Subunits Reveals Modes and Regulation of Translation Initiation.

Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5' UTRs and stop codon recognition, in a higher eukaryote.

Histidine triad nucleotide-binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A.

Human histidine triad nucleotide-binding protein 2 (hHINT2) is an important player in human mitochondrial bioenergetics, but little is known about its catalytic capabilities or its nucleotide phosphoramidate prodrug (proTide)-activating activity akin to the cytosolic isozyme hHINT1. Here, a similar substrate specificity profile (k /K ) for model phosphoramidate substrates was found for hHINT2 but with higher k and K values when compared with hHINT1. A broader pH range for maximum catalytic activity was determined for hHINT2 (pK  = 6.

Repression of oncogenic cap-mediated translation by 4Ei-10 diminishes proliferation, enhances chemosensitivity and alters expression of malignancy-related proteins in mesothelioma.

Activated cap-dependent translation promotes cancer by stimulating translation of mRNAs encoding malignancy-promoting proteins. The nucleoside monophosphate Protide, 4Ei-10, undergoes intracellular uptake and conversion by Hint1 to form 7-Cl-Ph-Ethyl-GMP. 7-Cl-Ph-Ethyl-GMP is an analog of cap and inhibits protein translation by binding and sequestering eIF4E thus blocking eIF4E from binding to the mRNA cap.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females.

Inhibition of HINT1 Modulates Spinal Nociception and NMDA Evoked Behavior in Mice.

The interactions between the mu-opioid (MOR) and -methyl-d-aspartate receptor (NMDAR) constitute an area of intense investigation because of their contributions to maladaptive neuroplasticity. Recent evidence suggests that their association requires the involvement of histidine triad nucleotide-binding protein (HINT1) with the enzyme's active site being critical in its regulatory role. Since it is known that spinal blockade of NMDA receptors prevents the development of opioid analgesic tolerance, we hypothesized that spinal inhibition of the HINT1 enzyme may similarly inhibit opioid tolerance.

Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure.

Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral neuropathy following minor nerve injury. This effect coincides with elevated spinal cord astrocyte activation and ex vivo immune cell reactivity assessed by proinflammatory cytokine interleukin (IL) -1β protein expression. Additionally, the β2-integrin adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1), a factor that influences the expression of the proinflammatory/anti-inflammatory cytokine network is upregulated.

In-DANBIRT Molecular Imaging of Inflammatory Cells in Atherosclerosis.

Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications.