Polymer coatings are widely used to suppress nonspecific protein adsorption and modulate nanoparticle-cell interactions. However, in complex biological systems, the biological outcomes of polymer-functionalized nanoparticles often deviate from their intended design owing to rapid protein corona formation that alters nanoparticle identity. Compounding this challenge, there is currently no unified platform for systematic evaluation of polymer coatings under comparable surface functionalization and biologically relevant conditions. To address this limitation, we establish a modular grafting-to platform based on cellulose nanocrystals (CNCs) that enables systematic comparison of polyethylene glycol (PEG)-based, zwitterionic, and acrylamide-based polymers with independent control over grafting density and chain conformation. Using this CNC- based model system, we demonstrate that dense polymer brushes reduce nonspecific protein adsorption in single-protein assays. By contrast, all CNC surfaces form albumin-rich coronas upon exposure to complex mixtures of serum proteins, although specific polymer coatings selectively enrich or suppress distinct protein populations. We further demonstrate that polymer-dependent immune cell interactions observed under serum-free conditions are largely attenuated following protein corona formation. Nevertheless, differences among polymer chemistries persist, with zwitterionic coatings broadly suppressing immune cell interactions and PEG-based coatings selectively promoting interactions with antigen-presenting cells, such as B cells and conventional dendritic cells. These findings establish design principles for polymer-functionalized nanoparticles with defined immune cell interaction profiles.

Thyroid hormone (TH) signaling plays a major role in the development, energy homeostasis, and metabolism of most tissues. Recent observations have identified THs as drivers of prostate cancer (PCa) tumor development and progression. We reported that the T3-scavenger protein µ-crystallin (CRYM) regulates the development and progression of PCa and that this involved crosstalk with the androgen receptor (AR) signaling. However, the mechanisms remain incompletely understood. Here, we explored the role of thyroid hormone receptor β (TRβ), which is the main effector of TH signaling, in the context of PCa. The use of the TRβ-selective antagonist NH-3 inhibited PCa cell proliferation in vitro and reduced tumor size in PCa xenograft models. Notably, NH-3 was highly effective in the engrafted 22Rv1 cell line, a model for castration-resistant PCa (CRPC). Mechanistic studies revealed that NH-3 downregulates AR and the AR target genes Nkx3.1 and KLK3 ( PSA ). NH-3 was a more effective anticancer agent than enzalutamide and showed synergistic properties in combined use. Evidence from human datasets corroborates our findings whereby elevated TRβ expression and mutations in TH signaling pathways are associated with the onset of PCa. Collectively, these results establish TRβ as a mediator of tumorigenesis in PCa and identify NH-3 as a promising therapeutic agent for targeting AR signaling, particularly in CRPC.

Nerve injury triggers localized translation of axonal mRNAs to respond to injury and nerve regeneration. The core stress granule protein G3BP1 sequesters axonal mRNAs in granules before and after axotomy. G3BP1 granule disassembly can be regulated by post-translational modifications, including phosphorylation of S149 phosphorylation and acetylation of human K376 (mouse K374). Axonal G3BP1 undergoes phosphorylation after axotomy, but acetylation of G3BP1 in axons was unknown. Here we show that rodent G3BP1 undergoes K374 acetylation after axotomy is ELP3-dependent, which enhances axonal protein synthesis, accelerates nerve regeneration, and supports functional recovery. ELP3-depleted neurons exhibit reduced axon growth and increased axonal G3BP1 granules. The proximal axons degenerate rapidly despite maintaining soma connectivity, an effect prevented by expression of acetylmimetic G3BP1.Together, these findings identify G3BP1 acetylation via ELP3 as a critical regulator of both axonal regeneration and neuronal resilience, revealing a post-translational mechanism that links stress granule regulation to neuronal repair and protection.

Hydroxamic acid (HA)-based HDAC inhibitors often suffer from poor pharmacokinetic (PK) profiles, limiting their in vivo applications. Cap group modification offers a promising strategy to address these challenges. Here, we optimized the cap group of TO-317, a selective HDAC6 inhibitor with a bisected cap structure, generating 26 analogs with comparable or improved HDAC6 binding affinity and selectivity. Replacing the redundant tetrafluorobenzene sulfonamide cap while retaining the essential picolyl cap group preserved the critical H614 hydrogen bond, as confirmed by X-ray crystallography (1.24-1.27 Å resolution) of five analogs. Analog 14, featuring a 2-chlorobenzene sulfonamide cap, demonstrated a 120-fold enhancement in plasma concentration in mice compared to that of TO-317. Preclinical studies showed that analog 14 achieved 56% tumor growth inhibition in an SM1 melanoma murine model without observed toxicity. These findings highlight cap group optimization as a powerful approach to enhance HA-based HDAC inhibitors for advanced preclinical and clinical development.

Herein, we report a potent HDAC6 PROTAC, TO-1187, which selectively degrades HDAC6 in cellulo and demonstrates in vivo efficacy. The design of TO-1187 was achieved by linking our previously reported HDAC6 inhibitor, TO-317, to the cereblon (CRBN) E3 ligase ligand, pomalidomide. TO-1187 achieved monoselective HDAC6 degradation in human multiple myeloma cells, MM.1S, with a Dmax of 94% and a DC50 of 5.81 nM after 6 h. Importantly, at concentrations up to 25 μM, TO-1187 exhibited no cellular degradation of other HDACs. Proteomic evaluation confirmed a highly selective proteome-wide degradation profile, with HDAC6 the only protein observed to be depleted. Notably, TO-1187 did not impact the abundance of well-known CRBN neosubstrates, like IKZF1, IKZF3, CK1α, SALL4, and GSPT1. In vivo evaluation confirmed that TO-1187 efficiently degraded HDAC6 in mouse tissues, measured 6 h after intravenous injection. In summary, TO-1187 represents a viable candidate for advanced preclinical evaluation of HDAC6 biology.

Targeted protein degradation (TPD) is revolutionizing drug discovery, but PROTAC synthesis remains challenging due to multi-step synthesis and slow linker installation via SNAr on 4-fluorothalidomide. Here, we optimize a microwave-assisted synthesis (MAS) of pomalidomide building blocks, achieving high yields within 15 min - boosting yield by at least 14% at gram scale without the need for purification. Unlike conventional oil bath heating and overnight reactions, MAS streamlines degrader development. The method's utility was demonstrated by synthesizing ARV-110, highlighting MAS as a powerful tool for accelerating pomalidomide PROTAC and molecular glue discovery programs.

Thyroid hormone (TH) signaling plays a major role in the development, energy homeostasis, and metabolism of most tissues. Recent studies have identified THs as drivers of prostate cancer (PCa) development and progression. We reported that the T3-scavenger protein µ-crystallin (CRYM) regulates the development and progression of PCa and that this involved crosstalk with androgen receptor (AR) signaling. However, the mechanisms remain incompletely understood. Here, we explored the role of thyroid hormone receptor β (TRβ), which is the main effector of TH signaling, in the context of PCa. The use of the TRβ-selective antagonist NH-3 inhibited PCa cell proliferation in vitro and reduced tumor size in PCa xenograft models in vivo. Notably, NH-3 was highly effective in the engrafted 22Rv1 cell line, a model for castration-resistant PCa (CRPC). Mechanistic studies revealed that NH-3 downregulates AR and the AR target genes Nkx3.1 and KLK3 (PSA). NH-3 was a more effective anticancer agent than enzalutamide, and their combined use was synergistic. Evidence from human datasets corroborates our findings, whereby elevated TRβ expression and mutations in the TH signaling pathway are associated with the onset of PCa. Collectively, these results establish TRβ as a mediator of tumorigenesis in PCa and identify NH-3 as a promising therapeutic agent for targeting AR signaling, particularly in CRPC.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Patients with brain metastases (BM) face a 90% mortality rate within one year of diagnosis and the current standard of care is palliative. Targeting BM-initiating cells (BMICs) is a feasible strategy to treat BM, but druggable targets are limited. Here, we apply Connectivity Map analysis to lung-, breast-, and melanoma-pre-metastatic BMIC gene expression signatures and identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a target for BM. We show that pharmacological and genetic perturbation of IMPDH attenuates BMIC proliferation in vitro and the formation of BM in vivo. Metabolomic analyses and CRISPR knockout studies confirm that de novo GTP synthesis is a potent metabolic vulnerability in BM. Overall, our work employs a phenotype-guided therapeutic strategy to uncover IMPDH as a relevant target for attenuating BM outgrowth, which may provide an alternative treatment strategy for patients who are otherwise limited to palliation.

The transcription factors STAT3, STAT5A, and STAT5B steer hematopoiesis and immunity, but their enhanced expression and activation promote acute myeloid leukemia (AML) or natural killer/T cell lymphoma (NKCL). Current therapeutic strategies focus on blocking upstream tyrosine kinases to inhibit STAT3/5, but these kinase blockers are not selective against STAT3/5 activation and frequent resistance causes relapse, emphasizing the need for targeted drugs. We evaluated the efficacy of JPX-0700 and JPX-0750 as dual STAT3/5 binding inhibitors promoting protein degradation. JPX-0700/-0750 decreased the mRNA and protein levels of STAT3/5 targets involved in cancer survival, metabolism, and cell cycle progression, exhibiting nanomolar to low micromolar efficacy. They induced cell death and growth arrest in both AML/NKCL cell lines and primary AML patient blasts. We found that both AML/NKCL cells hijack STAT3/5 signaling through either upstream activating mutations in kinases, activating mutations in STAT3, mutational loss of negative STAT regulators, or genetic gains in anti-apoptotic, pro-proliferative, or epigenetic-modifying STAT3/5 targets. This emphasizes a vicious cycle for proliferation and survival through STAT3/5. Both JPX-0700/-0750 treatment reduced leukemic cell growth in human AML or NKCL xenograft mouse models significantly, being well tolerated by mice. Synergistic cell death was induced upon combinatorial use with approved chemotherapeutics in AML/NKCL cells.

Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine (19F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied via fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large 19F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of 13C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl-13C6). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl-13C6) 5-fluorotryptophan. The resulting two-dimensional 19F-13C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The (19F,13C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs.

Fluorine NMR is a highly sensitive technique for delineating the conformational states of biomolecules and has shown great utility in drug screening and in understanding protein function. Current fluorinated protein tags leverage the intrinsic chemical shift sensitivity of the 19F nucleus to detect subtle changes in protein conformation and topology. This chemical shift sensitivity can be amplified by embedding the fluorine or trifluoromethyl reporter within a pyridone. Due to their polarizability and rapid tautomerization, pyridones exhibit a greater range of electron delocalization and correspondingly greater 19F NMR chemical shift dispersion. To assess the chemical shift sensitivity of these tautomeric probes to the local environment, 19F NMR spectra of all possible monofluorinated and trifluoromethyl-tagged versions of 2-pyridone were recorded in methanol/water mixtures ranging from 100% methanol to 100% water. 4-Fluoro-2-pyridone and 6-(trifluoromethyl)-2-pyridone (6-TFP) displayed the greatest sensitivity of the monofluorinated and trifluoromethylated pyridones, exceeding that of known conventional CF3 reporters. To evaluate the utility of tautomeric pyridone tags for 19F NMR of biomolecules, the alpha subunit of the stimulatory G protein (Gsα) and human serum albumin (HSA) were each labeled with a thiol-reactive derivative of 6-TFP and the spectra were recorded as a function of various adjuvants and drugs. The tautomeric tag outperformed the conventional tag, 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide through the improved resolution of several functional states.

Collectively known as psoriatic disease, psoriasis and psoriatic arthritis (PsA) are immune-mediated inflammatory diseases in which patients present with cutaneous and musculoskeletal inflammation. Affecting roughly 2-3% of the world's total population, there remains unmet therapeutic needs in both psoriasis and PsA despite the availability of current immunomodulatory treatments. As a result, patients with psoriatic disease often experience reduced quality of life. Recently, a class of small molecules, commonly investigated as anti-cancer agents, called histone deacetylase (HDAC) inhibitors, have been proposed as a new promising anti-inflammatory treatment for immune- and inflammatory-related diseases. In inflammatory diseases, current evidence is derived from studies on diseases like rheumatoid arthritis (RA) and systematic lupus erythematosus (SLE), and while there are some reports studying psoriasis, data on PsA patients are not yet available. In this review, we provide a brief overview of psoriatic disease, psoriasis, and PsA, as well as HDACs, and discuss the rationale behind the potential use of HDAC inhibitors in the management of persistent inflammation to suggest its possible use in psoriatic disease.

The oncogenic transcription factors STAT3, STAT5A and STAT5B are essential to steer hematopoiesis and immunity, but their enhanced expression and activation drives the development or progression of blood cancers. Current therapeutic strategies focus on blocking upstream tyrosine kinases, but frequently occurring resistance often leads to disease relapse, emphasizing the need for more targeted therapies. Here we evaluate JPX-0700 and JPX-0750, which are STAT3/5-specific covalent cysteine binders that lead to growth arrest of acute myeloid leukemia (AML) and natural killer/T cell lymphoma (NKCL) cell lines in vitro and in vivo , as well as reduce cell viability of primary AML blasts ex vivo . Our non-PROTAC small molecular weight degraders selectively reduce STAT3/5 activation and total protein levels, as well as downstream target oncogene expression, exhibiting nanomolar to low micromolar efficacy. We found that both AML and NKCL cells hijack STAT3/5 signaling through either upstream activating mutations in tyrosine kinases, activating gain-of-function mutations in STAT3, mutational loss of negative STAT regulators, or genetic gains in anti-apoptotic, pro-proliferative or epigenetic-modifying STAT3/5 targets. Moreover, we have shown synergistic inhibitory action of JPX-0700 and JPX-0750 upon combinatorial use with approved chemotherapeutics (doxorubicin, daunorubicin, cytarabine), epigenetic enzyme blocker vorinostat, tyrosine kinase inhibitor cabozantinib or BCL-2 inhibitor venetoclax. Importantly, JPX-0700 or JPX-0750 treatment reduced leukemic cell growth in human AML/NKCL xenograft mouse models without adverse side effects. These potent small molecule degraders of STAT3/5 could propel further clinical development for use in AML and NKCL patients.

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of epsilon-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2(Cys274) (catalytic domain), with nM potency against HDAC1-3, sub-mu M activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Lanthanide-doped upconversion nanoparticles (UCNPs) possess the remarkable ability to convert multiple near-infrared (NIR) photons into higher energy ultraviolet-visible (UV-vis) photons, making them a prime candidate for several advanced applications within the realm of nanotechnology. Compared to traditional organic fluorophores and quantum dots (QDs), UCNPs possess narrower emission bands (fwhm of 10-50 nm), large anti-Stokes shifts, low toxicity, high chemical stability, and resistance to photobleaching and blinking. In addition, unlike UV-vis excitation, NIR excitation is nondestructive at lower power intensities and has high tissue penetration depths (up to 2 mm) with low autofluorescence and scattering. Together, these properties make UCNPs exceedingly favored for advanced bioanalytical and theranostic applications, where these systems have been well-explored. UCNPs are also well-suited for bioimaging, optically modulating chemistries, forensic science, and other state-of-the-art research applications. In this review, an up-to-date account of emerging applications in UCNP research, beyond bioanalytical and theranostics, are presented including optogenetics, super-resolution imaging, encoded barcodes, fingerprinting, NIR vision, UCNP-assisted photochemical manipulations, optical tweezers, 3D printing, lasing, NIR-II imaging, UCNP-molecule nanohybrids, and UCNP-based persistent luminescent nanocrystals.

Supplementary Figure S1. Stat5 expression correlates with the expression of anti-apoptotic genes. Supplementary Figure S2. STAT5-deficient NK cells possess aberrant transcription factors expression. Supplementary Figure S3. Loss of STAT5 alters granzyme, perforin and IFN-gamma production. Supplementary Figure S4. STAT5-deficient NK cells induce tumor promotion. Supplementary Figure S5. Loss of STAT5 increases expression of the pro-angiogenic factor VEGF-A. Supplementary Figure S6. Peripheral splenic NK cells produce VEGF-A which does not impact NK cell numbers, proliferation or maturation but enhances tumor progression in lymphoid tumor models. Supplementary Figure S7. Tumor-infiltrating NK cells and macrophages are localized around blood vessels. Supplementary Figure S8. IL-2 stimulated human NK cells mirror the murine data.

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.

Histone deacetylases (HDACs) are a class of zinc (Zn)-dependent metalloenzymes that are responsible for epigenetic modifications. HDACs are largely associated with histone proteins that regulate gene expression at the DNA level. This tight regulation is controlled by acetylation [via histone acetyl transferases (HATs)] and deacetylation (via HDACs) of histone and non-histone proteins that alter the coiling state of DNA, thus impacting gene expression as a downstream effect. For the last two decades, HDACs have been studied extensively and indicated in a range of diseases where HDAC dysregulation has been strongly correlated with disease emergence and progression—most prominently, cancer, neurodegenerative diseases, HIV, and inflammatory diseases. The involvement of HDACs as regulators in these biochemical pathways established them as an attractive therapeutic target. This review summarizes the drug development efforts exerted to create HDAC inhibitors (HDACis), specifically class I HDACs, with a focus on the medicinal chemistry, structural design, and pharmacology aspects of these inhibitors.

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.