Discovery of a potent and selective JAK1-targeting PROTAC degrader with anti-tumor activities.

Aberrant activation of the JAK-STAT pathway is evident in various human diseases including cancers. Proteolysis targeting chimeras (PROTACs) provide an attractive strategy for developing novel JAK-targeting drugs. Herein, a series of CRBN-directed JAK-targeting PROTACs were designed and synthesized utilizing a JAK1/JAK2 dual inhibitor-momelotinib as the warhead. The most promising compound 10c exhibited both good enzymatic potency and cellular antiproliferative effects. Western blot analysis revealed that compound 10c effectively and selectively degraded JAK1 in a proteasome-dependent manner (DC50 = 214 nM). Moreover, PROTAC 10c significantly suppressed JAK1 and its key downstream signaling. Together, compound 10c may serve as a novel lead compound for antitumor drug discovery.

Drugtamer-PROTAC Conjugation Strategy for Targeted PROTAC Delivery and Synergistic Antitumor Therapy.

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising strategy for targeted protein degradation and drug discovery. To overcome the inherent limitations of conventional PROTACs, an innovative drugtamer-PROTAC conjugation approach is developed to enhance tumor targeting and antitumor potency. Specifically, a smart prodrug is designed by conjugating "drugtamer" to a nicotinamide phosphoribosyltransferase (NAMPT) PROTAC using a tumor microenvironment responsible linker. The "drugtamer" consists of fluorouridine nucleotide and DNA-like oligomer. Compared to NAMPT PROTAC and the combination of PROTAC + fluorouracil, the designed prodrug AS-2F-NP demonstrates superior tumor targeting, efficient cellular uptake, improved in vivo potency and reduced side effects. This study provides a promising strategy for the precise delivery of PROTAC and synergistic antitumor agents.

The Shape Effect of Acoustic Micropillar Array Chips in Flexible Label-Free Separation of Cancer Cells.

The precise isolation of circulating tumor cells (CTCs) from blood samples is a potent tool for cancer diagnosis and clinical prognosis. However, CTCs are present in extremely low quantities in the bloodstream, posing a significant challenge to their isolation. In this study, we propose a non-contact acoustic micropillar array (AMPA) chip based on acoustic streaming for the flexible, label-free capture of cancer cells. Three shapes of micropillar array chips (circular, rhombus, and square) were fabricated. The acoustic streaming characteristics generated by the vibration of microstructures of different shapes are studied in depth by combining simulation and experiment. The critical parameters (voltage and flow rate) of the device were systematically investigated using microparticle experiments to optimize capture performance. Subsequently, the capture efficiencies of the three micropillar structures were experimentally evaluated using mouse whole blood samples containing cancer cells. The experimental results revealed that the rhombus microstructure was selected as the optimal shape, demonstrating high capture efficiency (93%) and cell activity (96%). Moreover, the reversibility of the acoustic streaming was harnessed for the flexible release and capture of cancer cells, facilitating optical detection and analysis. This work holds promise for applications in monitoring cancer metastasis, bio-detection, and beyond.

Design of Evodiamine-Glucose Conjugates with Improved In Vivo Antitumor Activity.

Natural product evodiamine is a multitargeting antitumor lead compound. However, clinical development of evodiamine derivatives was hampered by poor water solubility and limited in vivo antitumor potency. Herein, a series of evodiamine-glucose conjugates were designed by additional targeting glucose transporter-1 (GLUT1). Compared with the lead compound, conjugate 8 exhibited obvious enhancement in water solubility and in vivo antitumor efficacy. Furthermore, the effect of GLUT1 targeting also led to lower cytotoxicity to normal cells. Antitumor mechanism studies manifested that conjugate 8 acted by Top1/Top2 dual inhibition, apoptosis induction, and G2/M cell cycle arrest, which selectively targeted tumor cells with a high expression level of GLUT1. Thus, evodiamine-glucose conjugates showed promising features as potential antitumor agents.

The Association between Serum Copper and Bone Mineral Density among Adolescents Aged 12 to 19 in the United States.

Bone mineral density (BMD) is a key parameter widely used in the assessment of bone health. Although many investigations have explored the relationship between trace elements and BMD, there are fewer studies focused on serum copper and BMD, especially for adolescents. Using data extracted from the National Health and Nutrition Examination Survey, we applied a multiple-linear regression and smooth curve fitting to assess the relationship between serum copper and BMD. A total of 910 participants were finally included in this study. After adjusting for relevant covariates, serum copper was negatively associated with lumbar spine BMD (ß = -0.057, 95% CI: -0.109 to -0.005), trunk bone BMD (ß = -0.068, 95% CI: -0.110 to -0.026), pelvis BMD (ß = -0.085, 95% CI: -0.145 to -0.024), subtotal BMD (ß = -0.072, 95% CI: -0.111 to -0.033), and total BMD (ß = -0.051, 95% CI: -0.087 to -0.016) (p < 0.05). In quartile analysis, the highest level of serum copper was associated with decreased BMD when compared with those at the lowest quartile (p < 0.05). The stratified analysis revealed a significant interaction between age and the effects of serum copper on trunk bone BMD (p = 0.022) and pelvis BMD (p = 0.018). Meanwhile, the higher level of serum copper was negatively associated with BMD in males, and gender modified the relationship (p < 0.001). Future longitudinal studies will be necessary for a more definitive interpretation of our results.

Discovery of a Potent Nicotinamide Phosphoribosyltransferase Activator for Improving Aging-associated Dysfunctions.

Nicotinamide adenine dinucleotide (NAD+) plays a crucial role in the cellular energy metabolism pathway. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme involved in the biosynthesis of NAD+. Herein, a series of new NAMPT activators were designed to increase the NAD+ levels and improve aging-associated dysfunctions. In particular, compound C8 effectively activated NAMPT and promoted the biosynthesis of NAD+. Furthermore, we demonstrated that NAMPT activator C8 possessed excellent antiaging effects both in vitro and in vivo. Activator C8 showed potent activity in delaying aging in senescent HL-7702 cells and extended the lifespan of Caenorhabditis elegans. In a naturally aging mouse model, compound C8 effectively alleviated age-related dysfunctions and markers. Therefore, NAMPT activator C8 represented a promising lead compound for the treatment of age-related diseases.

Photoswitchable PROTACs for Reversible and Spatiotemporal Regulation of NAMPT and NAD.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme with diverse biological functions in DNA synthesis. Nicotinamide phosphoribosyltransferase (NAMPT) is a key rate-limiting enzyme involved in NAD+ biosynthesis in mammals. We developed the first chemical tool for optical control of NAMPT and NAD+ in biological systems using photoswitchable proteolysis-targeting chimeras (PS-PROTACs). An NAMPT activator and dimethylpyrazolazobenzene photoswitch were used to design highly efficient PS-PROTACs, enabling up- and down-reversible regulation of NAMPT and NAD+ in a light-dependent manner and reducing the toxicity associated with inhibitor-based PS-PROTACs. PS-PROTAC was activated under 620 nm irradiation, realizing in vivo optical manipulation of antitumor activity, NAMPT, and NAD+ .

Discovery of Novel PDEδ Autophagic Degraders: A Case Study of Autophagy-Tethering Compound (ATTEC).

The autophagy-tethering compound (ATTEC) technology has emerged as a promising strategy for targeted protein degradation (TPD). Here, we report the discovery of the first generation of PDEδ autophagic degraders using an ATTEC approach. The most promising compound 12c exhibited potent PDEδ binding affinity and efficiently induced PDEδ degradation in a concentration-dependent manner. Mechanistic studies confirmed that compound 12c reduced the PDEδ protein level through lysosome-mediated autophagy without affecting the PDEδ mRNA expression. Importantly, compound 12c was much more effective in suppressing the growth in KRAS mutant pancreatic cancer cells than the corresponding PDEδ inhibitor. Taken together, this study expands the application scope of the ATTEC approach and highlights the effectiveness of the PDEδ autophagic degradation strategy in antitumor drug discovery.

JAK1/JAK2 degraders based on PROTAC for topical treatment of atopic dermatitis.

Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disease. The Janus kinase (JAK) has been identified as a target in AD, as it regulates specific inflammatory genes and adaptive immune responses. However, the efficacy of topically applied JAK inhibitors in AD is limited due to the unique structure of skin. We synthesized JAK1/JAK2 degraders (JAPT) based on protein degradation targeting chimeras (PROTACs) and prepared them into topical preparations. JAPT exploited the E3 ligase to mediate ubiquitination and degradation of JAK1/JAK2, offering a promising AD therapeutic approach with low frequency and dosage. In vitro investigations demonstrated that JAPT effectively inhibited the release of pro-inflammatory cytokines and reduced inflammation by promoting the degradation of JAK. In vivo studies further confirmed the efficacy of JAPT in degrading JAK1/JAK2, leading to a significant suppression of type I, II, and III adaptive immunity. Additionally, JAPT demonstrated a remarkable reduction in AD severity, as evidenced by improved skin lesion clearance and AD severity scores (SCORAD). Our study revealed the therapeutic potential of JAPT, surpassing conventional JAK inhibitors in the treatment of AD, which suggested that JAPT could be a promising topically applied anti-AD drug targeting the JAK-STAT signaling pathway.

Dual-Programmable Semiconducting Polymer NanoPROTACs for Deep-Tissue Sonodynamic-Ferroptosis Activatable Immunotherapy.

Proteolysis-targeting chimeras (PROTACs) can provide promising opportunities for cancer treatment, while precise regulation of their activities remains challenging to achieve effective and safe therapeutic outcomes. A semiconducting polymer nanoPROTAC (SPNFeP ) is reported that can achieve ultrasound (US) and tumor microenvironment dual-programmable PROTAC activity for deep-tissue sonodynamic-ferroptosis activatable immunotherapy. SPNFeP is formed through a nano-precipitation of a sonodynamic semiconducting polymer, a ferroptosis inducer, and a newly synthesized PROTAC molecule. The semiconducting polymers work as sonosensitizers to produce singlet oxygen (1 O2 ) via sonodynamic effect under US irradiation, and ferroptosis inducers react with intratumoral hydrogen peroxide (H2 O2 ) to generate hydroxyl radical (·OH). Such a dual-programmable reactive oxygen species (ROS) generation not only triggers ferroptosis and immunogenic cell death (ICD), but also induces on-demand activatable delivery of PROTAC molecules into tumor sites. The effectively activated nanoPROTACs degrade nicotinamide phosphoribosyl transferase (NAMPT) to suppress tumor infiltration of myeloid-derived suppressive cells (MDSCs), thus promoting antitumor immunity. In such a way, SPNFeP mediates sonodynamic-ferroptosis activatable immunotherapy for entirely inhibiting tumor growths in both subcutaneous and 2-cm tissue-covered deep tumor mouse models. This study presents a dual-programmable activatable strategy based on PROTACs for effective and precise cancer combinational therapy.

Smart glypican-3-targeting peptide-chlorin e6 conjugates for targeted photodynamic therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive and lethal malignancy with poor prognosis, necessitating the urgent development of effective treatments. Targeted photodynamic therapy (PDT) offers a promising way to selectively eradicate tumor cells without affecting normal cells. Inspired by promising features of peptide-drug conjugates (PDCs) in targeted cancer therapy, herein a novel glypican-3 (GPC3)-targeting PDC-PDT strategy was developed for the precise PDT treatment of HCC. The GPC3-targeting photosensitizer conjugates were developed by attaching GPC3-targeting peptides to chlorin e6. Conjugate 8b demonstrated the ability to penetrate HCC cells via GPC3-mediated entry process, exhibiting remarkable tumor-targeting capacity, superior antitumor efficacy, and minimal toxicity towards normal cells. Notably, conjugate 8b achieved complete tumor elimination upon light illumination in a HepG2 xenograft model without harm to normal tissues. Overall, this innovative GPC3-targeting conjugation strategy demonstrates considerable promise for clinical applications for the treatment of HCC.

Discovery of Dual Function Agents That Exhibit Anticancer Activity via Catastrophic Nicotinamide Adenine Dinucleotide Depletion.

Nicotinamide adenine dinucleotide (NAD) is essentially involved in many biological processes of cancer cells, yet chemical intervention of NAD biosynthesis failed to obtain an optimal therapeutic benefit. We herein developed a new strategy to induce catastrophic NAD depletion by concurrently impairing NAD synthesis and promoting NAD consumption. We designed a series of new compounds that conjugate an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD salvage pathway, with a DNA-alkylating agent. Among them, compound 11b exhibited potent anticancer efficacy in cancer cell lines and mouse tumor models with intrinsic resistance to the parent compound FK866 or chlorambucil. Compound 11b caused catastrophic NAD depletion via a synergistic effect between the NAD salvage pathway blockade and DNA damage-triggered NAD consumption. Our findings suggest a new intervention strategy for causing catastrophic NAD depletion in cancer cells and provide basis for the development of new inhibitors targeting NAD metabolism.

Enhanced Tumor Targeting and Penetration of Proteolysis-Targeting Chimeras through iRGD Peptide Conjugation: A Strategy for Precise Protein Degradation in Breast Cancer.

Proteolysis-targeting chimeras (PROTACs) have recently emerged as a promising technology for drug development. However, poor water solubility, limited tissue selectivity, and inadequate tumor penetration pose significant challenges for PROTAC-based therapies in cancer treatment. Herein, we developed an iRGD-PROTAC conjugation strategy utilizing tumor-penetrating cyclic peptide iRGD (CRGDK/RGPD/EC) to deliver PROTACs deep into breast cancer tissues. As a conceptual validation study, iRGD peptides were conjugated with a bromodomain-containing protein 4 (BRD4) PROTAC through a GSH-responsive linker. The resulting iRGD-PROTAC conjugate iPR showed enhanced water solubility, tumor-targeting capability, and penetration within tumor tissues, resulting in increased antibreast cancer efficacy in animal models and patient-derived organoids. This study demonstrates the advantages of combining iRGD and PROTACs in improving drug delivery and highlights the importance of tissue selectivity and penetration ability in PROTAC-based therapeutics.

PROTAC derivatization of natural products for target identification and drug discovery: Design of evodiamine-based PROTACs as novel REXO4 degraders.

INTRODUCTION: Natural products (NPs) play a crucial role in the development of therapeutic drugs. However, it is still highly challenging to identify the targets of NPs. Besides, NPs usually exert their pharmacological activities via acting on multiple targets or pathways, which also poses great difficulties for the target identification of NPs. OBJECTIVES: Inspired by our continuous efforts in designing drug-like protein degraders, this study introduced a successful example for the target identification and drug discovery of natural products evodiamine by employing PROTAC technology. METHODS: Taking advantages of proteolysis targeting chimera (PROTAC), herein an integrated strategy combining PROTAC derivatization, quantitative proteomic analysis and binding affinity validation was developed for target identification and drug discovery of antitumor NP evodiamine. RESULTS: In this study, both highly potent PROTACs and negative controls were designed for quantitative proteomic analysis. Furthermore, REXO4 was confirmed as a direct target of 3-fluoro-10-hydroxylevodiamine, which induced cell death through ROS. In addition, the PROTAC 13c effectively degraded REXO4 both in vitro and in vivo, leading to potent antitumor activities and reduced toxic side effects. CONCLUSION: In summary, we developed an integrated strategy for the target identification and drug discovery of NPs, which was successfully applied to the PROTAC derivatization and target characterization of evodiamine. This proof-of-concept study highlighted the superiority of PROTAC technology in target identification of NPs and accelerated the process of NPs-based drug discovery, exhibiting broad application in NP-based drug development.

Grassland health assessment based on indicators monitored by UAVs: a case study at a household scale.

Grassland health assessment (GHA) is a bridge of study and management of grassland ecosystem. However, there is no standardized quantitative indicators and long-term monitor methods for GHA at a large scale, which may hinder theoretical study and practical application of GHA. In this study, along with previous concept and practices (i.e., CVOR, the integrated indexes of condition, vigor, organization and resilience), we proposed an assessment system based on the indicators monitored by unmanned aerial vehicles (UAVs)-UAVCVOR, and tested the feasibility of UAVCVOR at typical household pastures on the Qinghai-Tibetan Plateau, China. Our findings show that: (1) the key indicators of GHA could be measured directly or represented by the relative counterpart indicators that monitored by UAVs, (2) there was a significantly linear relationship between CVOR estimated by field- and UAV-based data, and (3) the CVOR decreased along with the increasing grazing intensity nonlinearly, and there are similar tendencies of CVOR that estimated by the two methods. These findings suggest that UAVs is suitable for GHA efficiently and correctly, which will be useful for the protection and sustainable management of grasslands.

Discovery of potent NAMPT-Targeting PROTACs using FK866 as the warhead.

Nicotinamide phosphoribosyltransferase (NAMPT) has emerged as a promising target for cancer therapy due to its strong correlation with nicotinamide adenine dinucleotide (NAD+) metabolism and tumorigenesis. Proteolysis targeting chimeras (PROTACs) provided an attractive strategy for developing NAMPT-targeting NAD+-depleting cancer drugs. Herein, a series of von Hippel-Lindau (VHL)-recruiting NAMPT-targeting PROTACs were designed using NAMPT inhibitor FK866 as the warhead. Among them, compound C5 degraded NAMPT (DC50 = 31.7 nM) in a VHL- and proteasome-dependent manner. Moreover, compound C5 effectively inhibited the proliferation of A2780 cells (IC50 = 30.6 nM) and significantly reduced the general cytotoxicity of FK866 to normal cells.

Fluorescent and theranostic probes for imaging nicotinamide phosphoribosyl transferase (NAMPT).

Nicotinamide phosphoribosyl transferase (NAMPT) has been regarded as an attractive target for cancer therapy. However, there is a lack of chemical tools for real-time visualization and detection of NAMPT. Herein, the first fluorescent and theranostic probes were designed for imaging NAMPT, which had dual functions of diagnosis and treatment. The designed probes possessed good affinity and environmental sensitivity to NAMPT with a turn-on mechanism and were successfully applied in fluorescence detecting and imaging of NAMPT at the level of living cells and tissue sections. They also effectively inhibited tumor cell proliferation and arrested cell cycle at the G2 phase. These fluorescent probes enabled detection and visualization of NAMPT, representing effective chemical tools for the pathological diagnosis and treatment of cancer.

Discovery of Highly Potent Nicotinamide Phosphoribosyltransferase Degraders for Efficient Treatment of Ovarian Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is identified as a promising target for cancer therapy. However, known NAMPT inhibitors are characterized by weak clinical efficacy and dose-dependent toxicity. There is an urgent need to develop new NAMPT intervention strategies. Using the proteolysis-targeting chimera (PROTAC) technology, we designed and synthesized a series of new von Hippel-Lindau (VHL)-recruiting NAMPT-targeting PROTACs. A highly potent NAMPT degrader (B3) was successfully identified, which displayed excellent degradation activity (DC50 < 0.17 nM, Dmax > 90%) and antiproliferative potency against A2780 cells (IC50 = 1.5 nM). PROTAC B3 induced NAMPT depletion in a concentration- and time-dependent manner through the ubiquitin-proteasome system. Particularly, PROTAC B3 achieved good plasma exposure levels via intravenous injection, gained potent tumor growth inhibition (TGI = 88.1%, 2 µM/kg) in the xenograft model, and demonstrated good biosafety without undesired toxicities. This study provides a highly potent VHL-recruiting NAMPT degrader for the treatment of ovarian cancer.

Making Protein Degradation Visible: Discovery of Theranostic PROTACs for Detecting and Degrading NAMPT.

Proteolysis-targeting chimera (PROTAC) is emerging as a promising technology in targeted protein degradation and drug discovery. However, there is still a lack of effective chemical tools to real-time detect and track the protein degradation. Herein, the first fluorescent and theranostic PROTACs were designed for imaging the degradation of nicotinamide phosphoribosyltransferase (NAMPT) in living cells. Compound B4 was proven to be an environmentally sensitive fluorescent PROTAC, which efficiently degraded NAMPT (DC50 = 8.4 nM) and enabled the visualization of degradation in A2780 cells. As a theranostic agent, PROTAC B4 led to significant reduction of nicotinamide adenine dinucleotide (NAD+) and exerted potent antitumor activities both in vitro and in vivo. Collectively, this proof-of-concept study provides a new strategy for the real-time visualization of the process of protein degradation and the improvement of diagnosis and therapeutic efficacy of PROTACs.

Blocking Non-enzymatic Functions by PROTAC-Mediated Targeted Protein Degradation.

The non-enzymatic functions of target proteins play key roles in the regulation of various cell signaling pathways and are closely related to numerous human diseases. However, traditional small-molecule inhibitors generally target the catalytic functional domain directly and work by inhibiting the enzymatic function of the target proteins without affecting the non-enzymatic function. The recently emerging proteolysis targeting chimera (PROTAC) technology has the advantage of simultaneously regulating the enzymatic and non-enzymatic functions of target proteins, thus providing a potential strategy to make up for the deficiency of inhibitors and explore the new therapeutic profile by the target degradation. This perspective aims to specifically summarize and analyze recent progress in blocking non-enzymatic functions of target proteins by PROTAC-mediated degradation, highlighting representative case studies and discussing the pharmacological features originating from inhibition of the non-enzymatic functions.