Design, synthesis and biological evaluation of the positional isomers of the galactose conjugates able to target hepatocellular carcinoma cells via ASGPR-mediated cellular uptake and cytotoxicity.

Galactose as a recognizing motif for asialoglycoprotein receptor (ASGPR) is a widely accepted vector to deliver cytotoxic agents in the therapy of hepatocellular carcinoma (HCC), however, the individual hydroxyl group of galactose (Gal) contributed to recognizing ASGPR is obscure and remains largely unanswered in the design of glycoconjugates. Herein, we designed and synthesized five positional isomers of Gal-anthocyanin Cy5.0 conjugates and three Gal-doxorubicin (Dox) isomers, respectively. The fluorescence intensity of Gal-Cy5.0 conjugates accumulated in cancer cells hinted the optimal modification sites of positions C2 and C6. Comparing to the cytotoxicity of other conjugates, C2-Gal-Dox (11) was the most potent. Moreover, Gal-Dox conjugates significantly the toxicity of Dox. A progressively lower internalization capacity and siRNA technology implied the cellular uptake and cytotoxicity directly related to the ASGPR expression level. Accordingly, position C2 of galactose may be the best substitution site via ASGPR mediation in the design of anti-HCC glycoconjugates.

Assessment of Purity, Stability, and Pharmacokinetics of NGP-1, a Novel Prodrug of GS441254 with Potential Anti-SARS-CoV-2 Activity, Using Liquid Chromatography.

SARS-CoV-2 is a highly contagious and pathogenic virus that first appeared in late December 2019 and caused a global pandemic in a short period. The virus is a single-stranded RNA virus belonging to the Coronaviridae family. Numerous treatments have been developed and tested in response to the pandemic, particularly antiviral drugs. Among them, GS441524 (GS441), a nucleoside antiviral drug, has demonstrated promising results in inhibiting SARS-CoV-2. Nevertheless, the limited oral bioavailability of GS441 restricts its application to patients with the virus. In this study, a novel prodrug of GS441 (NGP-1) with an isobutyl ester and cyclic carbonate structure was designed and synthesized. Its purity and the stability in different artificial digestive juices of NGP-1 was determined with HPLC-DAD methods. The pharmacokinetics of NGP-1 and GS441 were studied in rats via gavage administration. A new LC-MS/MS method was developed to quantitatively analyze GS441 in plasma samples. The results showed that the ka, Cmax, and MRT of converted GS441 from NGP-1 were 5.9, 3, and 2.5 times greater than those of GS441 alone. The Frel of NGP-1 was approximately four-fold that of GS441, with an AUC0-∞ of 9716.3 h·ng mL-1. As a prodrug of GS441, NGP-1 increased its lipophilicity, absorption, and bioavailability, indicating that it holds promise in improving the clinical efficacy of anti-SARS-CoV-2 medications.

Discovery of new tetrahydroisoquinolines as potent and selective LSD1 inhibitors for the treatment of MLL-rearranged leukemia.

Histone lysine-specific demethylase 1 (LSD1) is a promising target for cancer therapy. Here, we performed the design, synthesis, and extensive structure-activity relationship (SAR) studies based on our previously discovered natural LSD1 inhibitor, higenamine. We found that the tetracyclic tetrahydroisoquinoline FY-21 is a potent and selective inhibitor of LSD1 (IC50 = 340 nM). FY-21 inhibited leukemia cell proliferation and colony formation and increased the level of p53 expression. Meanwhile, FY-21 reduced the mRNA levels of the transcription factors HOXA9 and MEIS1. Furthermore, FY-21 significantly induced leukemia cell differentiation. In vivo studies showed that FY-21 prolonged the survival rate of leukemia mice. Collectively, FY-21 is a potent, selective LSD1 inhibitor and can serve as a lead compound for the development of novel and highly effective LSD1 inhibitors for AML treatment.

Design, synthesis and immunological evaluation of monophosphoryl lipid A derivatives as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine.

Toll-like receptor 4 (TLR4) is a reliable target for the development of vaccine adjuvants. To identify novel TLR4 ligands with improved immunological properties for use as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine, herein, natural E. coli monophosphoryl lipid A (MPLA) and nine of its derivatives were designed and synthesized. Immunological evaluation showed that compounds 1, 3, 5 and 7 exhibited comparative or better adjuvant activity than clinically used Al adjuvants, and are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine. Preliminary structure-activity relationship analysis of the MPLA derivatives showed that the replacement of the functional groups at the C-1, C-4' or C-6' position of E. coli MPLA has an effect on its biological activity. In addition, we found that the combination of MPLA and Al was feasible for immunotherapy and could further enhance immune responses, providing a new direction toward the immunological enhancement of RBD-hFc based SARS-CoV-2 vaccines.

Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development.

We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule. Using Tn antigen as the model, a three-component vaccine (MPLA-Tn-KRN7000) containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized. This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers. The corresponding two-component conjugate vaccines Tn-MPLA, Tn-KRN7000 and Tn-CRM197 were also synthesized, as controls. The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity. The antibodies specifically recognized, bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells. In addition, MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice, and surviving mice reject further tumor attacks without any additional treatment. Compared to the glycoprotein vaccine Tn-CRM197, the two-component conjugate vaccines, Tn-MPLA and Tn-KRN7000, and the physical mixture of Tn-MPLA and Tn-KRN7000, MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both in vitro and in vivo. The comparison of immunological studies in wild-type and TLR4 knockout mice, along with the test of binding affinity to CD1d protein suggests that the covalently linked MPLA-KRN7000 immunostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn. This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

Human Fc-Conjugated Receptor Binding Domain-Based Recombinant Subunit Vaccines with Short Linker Induce Potent Neutralizing Antibodies against Multiple SARS-CoV-2 Variants.

The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319-541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319-583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.

Discovery of natural product-like spirooxindole derivatives as highly potent and selective LSD1/KDM1A inhibitors for AML treatment.

Histone lysine specific demethylase 1 (LSD1) is a promising new therapeutic target for cancer therapy. Following the work on the discovery of natural LSD1 inhibitor higenamine, we herein performed further structure-based design, synthesis, and extensive structure-activity relationship (SAR) studies, affording structurally new spirooxindole derivatives. Particularly, FY-56 was identified to be a highly potent LSD1 inhibitor (IC50 = 42 nM) and showed high selectivity over monoamine oxidases (MAO-A/B). Mechanistic studies showed that FY-56 moderately inhibited the proliferation and clone formation of leukemia cells, induced H3K4me1/2 accumulation and p53 activation as well as reduced the mRNA levels of the transcription factors HOXA9 and MEIS1. Meanwhile, FY-56 induced differentiation of MOLM-13 and MV4-11 cells, accompanied by an enhanced percentage of markers characteristic to differentiated macrophages and monocytes. Further in vivo studies showed that FY-56 obviously reduced the proportion of CD45+/CD33+ leukocytes in peripheral blood and spleen, and significantly prolonged the survival rate of mice. Collectively, FY-56 represents a structurally novel, highly potent and selective LSD1 inhibitor and exhibits therapeutic promise for AML treatment. The spirooxindole scaffold derived from FY-56 could be used to design structurally new LSD1 inhibitors for treating human diseases.

Ras inhibitor farnesylthiosalicylic acid conjugated with IR783 dye exhibits improved tumor-targeting and altered anti-breast cancer mechanisms in mice.

Ras has long been viewed as a promising target for cancer therapy. Farnesylthiosalicylic acid (FTS), as the only Ras inhibitor has ever entered phase II clinical trials, has yielded disappointing results due to its strong hydrophobicity, poor tumor-targeting capacity, and low therapeutic efficiency. Thus, enhancing hydrophilicity and tumor-targeting capacity of FTS for improving its therapeutic efficacy is of great significance. In this study we conjugated FTS with a cancer-targeting small molecule dye IR783 and characterized the anticancer properties of the conjugate FTS-IR783. We showed that IR783 conjugation greatly improved the hydrophilicity, tumor-targeting and therapeutic potential of FTS. After a single oral administration in Balb/c mice, the relative bioavailability of FTS-IR783 was increased by 90.7% compared with FTS. We demonstrated that organic anion transporting polypeptide (OATP) and endocytosis synergistically drove the uptake of the FTS-IR783 conjugate in breast cancer MDA-MB-231 cells, resulting in superior tumor-targeting ability of the conjugate both in vitro and in vivo. We further revealed that FTS-IR783 conjugate could bind with and directly activate AMPK rather than affecting Ras, and subsequently regulate the TSC2/mTOR signaling pathway, thus achieving 2-10-fold increased anti-cancer therapeutic efficacy against 6 human breast cancer cell lines compared to FTS both in vivo and in vitro. Overall, our data highlights a promising approach for the modification of the anti-tumor drug FTS using IR783 and makes it possible to return FTS back to the clinic with a better efficacy.

Discovery of higenamine as a potent, selective and cellular active natural LSD1 inhibitor for MLL-rearranged leukemia therapy.

Natural products are a rich source of lead compounds and have shown promise for epigenetic drug discovery. In this work, we discovered higenamine from our natural product library as a potent, selective and cellular active natural LSD1 inhibitor. Higenamine shows acceptable potency against LSD1 and high selectivity towards LSD1 over MAOA/B. Higenamine significantly increases expression of LSD1 substrates H3K4me1 and H3K4me2 in MLL-rearranged leukemia cells MV4-11 and MOLM-13, but nearly had no effect on LSD1 and H3K4Me3. Meanwhile, higenamine dose-dependently suppresses the levels of HOXA9 and MEIS1 that are overexpressed in leukemia cell lines. Notably, higenamine induces cell differentiation of MV4-11 and MOLM-13 cells accompanying by increased expression of CD11b, CD14 and CD86. Higenamine promotes cell apoptosis, inhibits colony formation, but does not inhibit proliferation of leukemia cells significantly. In addition, the expression levels of p53 are dramatically changed by higenamine in an LSD1-dependent manner in MV4-11 cells. Taken together, higenamine could be employed as a starting point for the development of more selective and potent LSD1 inhibitors. Our work firstly reveals the non-classical epigenetic regulation mechanism of higenamine in cancers, and also demonstrates the efficacy of higenamine for MLL-rearranged leukemia therapy.

Pubescenosides Q-R, two new phenolic glycosides from Ilex pubescens.

Two new phenolic glycosides (1-2), along with six existing compounds (3-8), were isolated from the ethanolic extract of Ilex pubescens roots, a traditional folk medicine. These structures were determined using HR-ESI-MS, IR, UV, and NMR (including 1 D, 2 D-NMR). The anti-inflammatory activities of three phenolic glycosides (1-3) were evaluated in the human HepG2 cell lines. The results showed that compound 3 could induce P-gp and BCRP expression through the Nrf2-mediated pathway.[Formula: see text].

Fully synthetic Mincle-dependent self-adjuvanting cancer vaccines elicit robust humoral and T cell-dependent immune responses and protect mice from tumor development.

A new strategy based on a macrophage-inducible C-type lectin (Mincle) agonist was established to construct synthetic cancer vaccines. Using sialyl-Tn (STn) as a model antigen, four conjugates with the Mincle agonist as a built-in adjuvant were designed and synthesized through a facile and efficient method. All conjugates could induce BMDMs to produce inflammatory cytokines in a Mincle-dependent manner and were found to elicit robust humoral and T cell-dependent immune responses alone in mice. The corresponding antibodies could recognize, bind and exhibit complement-dependent cytotoxicity to STn-positive cancer cells, leading to tumor cell lysis. Moreover, all conjugates could effectively inhibit tumor growth and prolong the mice survival time in vivo, with therapeutic effects better than STn-CRM197/Al. Notably, compared to conventional glycoprotein conjugate vaccines, these fully synthetic conjugate vaccines do not cause "epitope suppression." Mincle ligands thus hold great potential as a platform for the development of new vaccine carriers with self-adjuvanting properties for cancer treatment. Preliminary structure-activity relationship analysis shows that a vaccine containing one STn antigen carried by vizantin exhibits the best efficacy, providing support for further optimization and additional investigation into Mincle agonists as the carrier of self-adjuvanting cancer vaccines.

Small molecule inhibitors of the prostate cancer target KMT2D.

Histone lysine N-methyltransferase 2D (KMT2D), an important methyltransferase that is involved in the methylation of lysine 4 in histone H3 (H3K4) and related to the development of prostate cancer. Hypermethylation of H3K4 is shown in prostate cancer (PCa). However, KMT2D inhibitors have not yet been developed. This article aims to design small molecule inhibitors targeting KMT2D_SET to prevent PCa cell proliferation and migration. Twenty-four inhibitors were firstly designed according to a virtual screening of computers，and shown different degrees of binding to KMT2D_SET. Compounds 1 and 16 showed high binding affinities to KMT2D, with KD values of 147 ± 32.9 µM and 176 ± 37.9 µM, respectively. In addition, they exerted strong inhibitory activity against the PCa cell lines PC-3 and DU145, with IC50 values of 1.1 ± 0.06 µM, 1.5 ± 0.06 µM and 1.8 ± 0.1 µM, 2.3 ± 0.2 µM, respectively. Furthermore, these two compounds significantly suppressed the migration of PCa cells.

Small-molecule MDM2/X inhibitors and PROTAC degraders for cancer therapy: advances and perspectives.

Blocking the MDM2/X-P53 protein-protein interaction has been widely recognized as an attractive therapeutic strategy for the treatment of cancers. Numerous small-molecule MDM2 inhibitors have been reported since the release of the structure of the MDM2-P53 interaction in 1996, SAR405838, NVP-CGM097, MK-8242, RG7112, RG7388, DS-3032b, and AMG232 currently undergo clinical evaluation for cancer therapy. This review is intended to provide a comprehensive and updated overview of MDM2 inhibitors and proteolysis targeting chimera (PROTAC) degraders with a particular focus on how these inhibitors or degraders are identified from starting points, strategies employed, structure-activity relationship (SAR) studies, binding modes or co-crystal structures, biochemical data, mechanistic studies, and preclinical/clinical studies. Moreover, we briefly discuss the challenges of designing MDM2/X inhibitors for cancer therapy such as dual MDM2/X inhibition, acquired resistance and toxicity of P53 activation as well as future directions.

Natural products as LSD1 inhibitors for cancer therapy.

Natural products generally fall into the biologically relevant chemical space and always possess novel biological activities, thus making them a rich source of lead compounds for new drug discovery. With the recent technological advances, natural product-based drug discovery is now reaching a new era. Natural products have also shown promise in epigenetic drug discovery, some of them have advanced into clinical trials or are presently being used in clinic. The histone lysine specific demethylase 1 (LSD1), an important class of histone demethylases, has fundamental roles in the development of various pathological conditions. Targeting LSD1 has been recognized as a promising therapeutic option for cancer treatment. Notably, some natural products with different chemotypes including protoberberine alkaloids, flavones, polyphenols, and cyclic peptides have shown effectiveness against LSD1. These natural products provide novel scaffolds for developing new LSD1 inhibitors. In this review, we mainly discuss the identification of natural LSD1 inhibitors, analysis of the co-crystal structures of LSD1/natural product complex, antitumor activity and their modes of action. We also briefly discuss the challenges faced in this field. We believe this review will provide a landscape of natural LSD1 inhibitors.

Construction and immunogenic studies of a mFc fusion receptor binding domain (RBD) of spike protein as a subunit vaccine against SARS-CoV-2 infection.

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain (RBD, residues 319-541) and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice. The antibodies also effectively suppressed SARS-CoV-2 RBD binding to soluble ACE2, indicating that RBD-mFc may be further developed as a safe and effective SARS-CoV-2 vaccine.

Discovery of Novel Resorcinol Dibenzyl Ethers Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction as Potential Anticancer Agents.

Novel small molecule compounds based on various scaffolds including chalcone, flavonoid, and resorcinol dibenzyl ether were designed and tested for their inhibitory activity against the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 (PD-1/PD-L1) pathway. Among them, compound NP19 inhibited the human PD-1/PD-L1 interaction with IC50 values of 12.5 nM in homogeneous time-resolved fluorescence (HTRF) binding assays. In addition, NP19 dose-dependently elevated IFN-γ production in a coculture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, NP19 displayed significant in vivo antitumor efficacy in two different mouse models of cancer (a melanoma B16-F10 tumor model and an H22 hepatoma tumor model). Moreover, H&E staining and flow cytometry data suggested that NP19 activated the immune microenvironment in the tumor, which may contribute to its antitumor effects. This work shows NP19 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the PD-1/PD-L1 pathway.

Induction of lipid droplets in THP-1 macrophages by multi-walled carbon nanotubes in a diameter-dependent manner: A transcriptomic study.

Exposure to multi-walled carbon nanotubes (MWCNTs) might induce lipid droplet (LD) biogenesis, but the roles of physicochemical properties of MWCNTs, as well as the mechanisms, remain poorly understood. In this study, we investigated lipid laden foam formation in THP-1 macrophages exposed to MWCNTs of different diameters, and attempted transcriptomic analysis to study the possible mechanisms. We observed diameter-dependent cytotoxicity, lipid accumulation and intracellular reactive oxygen species production that were more pronounced for MWCNTs with smaller diameters compared with those with larger diameters. However, more MWCNTs with larger diameters were retained in macrophages after 24 h exposure. One possible explanation for the inverse relationship between MWCNT bio-effects and internalization is that macrophages altered the expression of exocytotic genes to export toxic MWCNTs. Transcriptomic data showed that MWCNTs with smaller diameters more effectively altered the expression of genes related with cytotoxicity and lipid metabolism, and KEGG pathway analysis suggested that MWCNTs with smaller diameters activated peroxisome proliferator-activated receptor (PPAR) signalling pathway (map03320), leading to over-expression of perilipin 2, the surface proteins of LDs. Western blot confirmed that MWCNTs effectively promoted CD36, PPARγ and perilipin 2, key components in map03320. Moreover, inhibition of PPARγ by chemicals or siRNA significantly inhibited lipid accumulation induced by MWCNTs with smaller diameters, and perilipin 2 proteins in MWCNT-exposed macrophages could be decreased by PPARγ siRNA. In conclusion, the results of this study revealed the induction of LDs by MWCNTs in a diameter-dependent manner through the activation of PPAR signalling pathway.

Synthesis of biologically relevant steroidal spiro ß-lactams from dienamides through the cascade 4-endo N-cyclization/aerobic oxidation sequence.

The steroid nucleus and ß-lactam are prevalent in natural products and drug molecules, the compounds containing such fragments always possess diverse and interesting biological profiles. Presented here is an unprecedented cascade 4-endo N-cyclization/aerobic oxidation sequence that enables the synthesis of biologically relevant steroidal spiro ß-lactams from dienamides. Of note, two continuous quaternary chiral centers were constructed simultaneously in this process, and the title compounds bearing the OH and CN groups are highly functionalized, allowing for late-stage transformations for construction of diverse compound library. The protocol has several advantages such as mild reaction conditions and short reaction time, therefore could serve as a new strategy for synthesizing ß-lactams.

LSD1/KDM1A inhibitors in clinical trials: advances and prospects.

Histone demethylase LSD1 plays key roles during carcinogenesis, targeting LSD1 is becoming an emerging option for the treatment of cancers. Numerous LSD1 inhibitors have been reported to date, some of them such as TCP, ORY-1001, GSK-2879552, IMG-7289, INCB059872, CC-90011, and ORY-2001 currently undergo clinical assessment for cancer therapy, particularly for small lung cancer cells (SCLC) and acute myeloid leukemia (AML). This review is to provide a comprehensive overview of LSD1 inhibitors in clinical trials including molecular mechanistic studies, clinical efficacy, adverse drug reactions, and PD/PK studies and offer prospects in this field.