Site-specific Antibody-Nitric Oxide Conjugate HN02 Possesses Improved Antineoplastic and Safety Properties.

Antibody-drug conjugates (ADCs) combine the high specificity of antibodies with the cytotoxicity of payloads and have great potential in pan-cancer immunotherapy. However, the current payloads for clinical uses have limited the therapeutic window due to their uncontrollable off-site toxicity. There is unmet needs to develop more potent ADC payloads with better safety and efficacy profiles. Nitric oxide (NO) is a special molecule that has low toxicity itself, which can kill tumor cells effectively when highly concentrated, has broad application prospects. Previously, we prepared for the first time an antibody-nitric oxide conjugate (ANC)-HN01, which showed inhibitory activity against hepatocellular carcinoma. However, the random conjugation method made HN01 highly heterogeneous and unstable. Here, we used site-specific conjugation-based engineered cysteine sites (CL-V211C) of anti-CD24 antibody to prepare a second-generation ANC with a drug-to-antibody ratio of 2. The homogeneous ANC, HN02 was stable in human plasma, shown in vitro bystander effect to neighboring cells and antiproliferative activity to CD24-targeted tumor cells. Compared with HN01, HN02 significantly prolonged the survival of tumor-bearing mice. In summary, we developed a stable and homogeneous site-specific conjugated ANC, which showed good antitumor activity and improved safety profile both in vitro and in vivo. This study provides new insight into the development of next generation of ADC candidates.

Synthesis and anti-tumor activity evaluation of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives with phenyl sulfonamide groups as potent RSK2 inhibitors.

Ribosome S6 Protein Kinase 2 (RSK2) is involved in many signal pathways such as cell growth, proliferation, survival and migration in tumors. Also, RSK2 can phosphorylate YB-1, which induces the expression of tumor initiating cells, leading to poor prognosis of triple negative breast cancer. Herein, phenyl sulfonamide was introduced to a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives to obtain novel RSK2 inhibitors which were evaluated RSK2 inhibitory activity and proliferation inhibitory activity against MDA-MB-468. The newly introduced sulfonamide group was observed to form a hydrogen bond with target residue LEU-74 which played crucial role in activity. The results showed that most of compounds exhibited RSK2 enzyme inhibitory with IC50 up to 1.7 nM. Compound B1 exhibited the strongest MDA-MB-468 cell anti-proliferation activity (IC50 = 0.13 µM). The in vivo tumor growth inhibitory activities were evaluated with compounds B1-B3 in MDA-MB-468 xenograft model which gave up to 54.6% of TGI.

Development of Integrated Bioorthogonal Self-Catalyzed NO Donor/Platinum(IV) Prodrugs for Synergistical Intervention against Triple-Negative Breast Cancer.

The platinum(IV) prodrug strategy is attractive for the synergistic antitumor effect. High levels (>400 nM) of nitric oxide (NO) exert promising cancer inhibition effects via multiple mechanisms. Herein, we designed and synthesized a new group of integrated bioorthogonal self-catalyzed NO donor/Pt(IV) prodrugs bearing long alkyl chains to enhance the stability in circulation, while the cytoplasmic reductants trigger cascade activation to release Pt and NO in tumor cells. Specifically, compound 10c exhibited an improved stability, favorable pharmacokinetic properties (AUC(0-t) of 2210.10 h*ng/mL), potent anti-triple-negative breast cancer (TNBC) effects (71.08% tumor growth inhibition (TGI) against the MDA-MB-231 xenograft model), potent in vivo anti-TNBC lung metastasis activity, and acceptable low toxicity. Importantly, NO released from 10c leads to the S-nitrosation of metal transporters Atox1&ATP7a in TNBC cells, which increases the Pt retention and inhibits lysyl oxidase, generating synergistic tumoricidal and antimetastatic activity. These results may inspire further study on the synergistical therapy of Pt and NO for the treatment of TNBC.

1,3-Substituted ß-Carboline Derivatives as Potent Chemotherapy for the Treatment of Cystic Echinococcosis.

Echinococcosis is a global public health issue that generally occurs in areas with developed animal husbandry. In search of safe and effective therapeutic agents against echinococcosis, we designed and synthesized new 1,3-substituted ß-carboline derivatives based on harmine. Among them, compounds 1a, 1c, and 1e displayed potent inhibitory activity against Echinococcus granulosus in vitro, significantly better than albendazole and harmine. The morphological detection revealed that 1a, 1c, and 1e significantly changed the ultrastructure of Echinococcus granulosus protoscolices (PSCs). Furthermore, pharmacokinetic studies suggested that 1a possessed a better metabolic property. Encouragingly, 1a exhibited a highest cyst inhibition rate as 76.8% in vivo and did not display neurotoxicity in mice. Further mechanistic research illustrated that 1a has the potential to induce autophagy in PSCs, which may be responsible for the therapeutic effect of the drugs. Together, 1a could be a promising therapeutic agent against echinococcosis, warranting further study.

Novel Nitric Oxide Donor-Azole Conjugation Strategy for Efficient Treatment of Cryptococcus neoformans Infections.

Invasive fungal infections (IFIs) such as cryptococcal meningitis (CM) remain a serious health issue worldwide due to drug resistance closely related to biofilm formation. Unfortunately, available antifungal drugs with ideal safety and promising potency are still lacking; thus, the research of new candidate and therapeutic approach is urgently needed. As an important gas messenger molecule, nitric oxide (NO) shows vital inhibition on various microorganism biofilms. Hence, three series of novel NO-donating azole derivatives were designed and synthesized, and the in vitro antifungal activity as well as the mechanism of action was investigated. Among them, 3a and 3e displayed excellent antifungal activity against Cryptococcus neoformans and biofilm depending on the release of NO. Moreover, a more stable analogue 3h of 3a demonstrated markedly anti-CM effects via intranasal dropping, avoiding the first-pass effects and possessing a better brain permeability bypass blood-brain barrier. These results present a promising antifungal candidate and intranasal dropping approach for the treatment of CM, warranting further studies.

Tirapazamine combined with photodynamic therapy improves the efficacy of ABZSO nanoparticles on Echinococcosis granulosus via further enhancing "breaking-then-curing".

BACKGROUND: Photodynamic therapy (PDT) has a promising application prospect in Echinococcus granulosus (Egs), however, the hypoxic environment of Egs and the hypoxia associated with PDT will greatly limit its effects. As a hypoxic-activated pre-chemotherapeutic drug, tirapazamine (TPZ) can be only activated and produce cytotoxicity under hypoxia environment. Albendazole sulfoxide (ABZSO) is the first choice for the treatment of Egs. This study aimed to explore the effects of ABZSO nanoparticles (ABZSO NPs), TPZ combined with PDT on the activity of Egs in vitro and in vivo. METHODS: The Egs were divided into control, ABZSO NPs, ABZSO NPs + PDT, and ABZSO NPs + TPZ + PDT groups, and the viability of Egs was determined using methylene blue staining. Then, the ROS, LDH and ATP levels were measured using their corresponding assay kit, and H2AX and TopoI protein expression was detected by western blot. The morphology of Egs with different treatments was observed using hematoxylin eosin (HE) staining and scanning electron microscopy (SEM). After that, the in vivo efficacy of ABZSO NPs, TPZ and PDT on Egs was determined in a Egs infected mouse model. RESULTS: In vitro experiments showed that the combined treatment of TPZ, ABZSO NPs and PDT significantly inhibited Egs viability; and significantly increased ROS levels and LDH contents, while decreased ATP contents in Egs; as well as up-regulated H2AX and down-regulated TopoI protein expression. HE staining and SEM results showed that breaking-then-curing treatment seriously damaged the Egs wall. Additionally, in vivo experiments found that the combination of ABZSO NPs, PDT and TPZ had more serious calcification and damage of the wall structure of cysts. CONCLUSIONS: ABZSO NPs combined with TPZ and PDT has a better inhibitory effect on the growth of Egs in vitro and in vivo based on the strategy of "breaking-then-curing".

One Stone Two Birds: Redox-Sensitive Colocalized Delivery of Cisplatin and Nitric Oxide through Cascade Reactions.

Bio-orthogonal bond-cleavage reactions have been used in cancer therapy for improving the biological specificity of prodrug activation, but the spatiotemporal consistency of reactants is still a huge challenge. Although, in most cases, the cleavage catalysts and caged prodrugs are administrated separately, it is difficult to avoid the reactions in advance before they meet at the tumor site. Herein, we design and construct novel coordinative nanoparticles, integrating two prodrugs A and B as ligands and ferric ions as coordinative centers. After nanoparticles accumulated in tumor through passive targeting, inert Pt(IV) prodrug A is specifically and spontaneously reduced into active Pt(II) cisplatin, which acts as the cleavage catalyst to subsequently initiate the in situ bio-orthogonal depropargylation of B, that is, O 2-propargyl nitric oxide (NO) donor. The unique structure of coordinative nanoparticles ensures the spatiotemporal consistency of reactants (prodrugs A and B) and products (cytotoxic cisplatin and tumoricidal NO) for the bio-orthogonal bond-cleavage reaction, which leads to an improved synergistic therapeutic activity for triple-negative breast cancer (TNBC). This new concept of bio-orthogonal dual-prodrug coordinative nanoparticles may inspire further applications in bio-orthogonal chemistry and drug delivery for combination chemotherapy.

Protein S-Nitrosation: Biochemistry, Identification, Molecular Mechanisms, and Therapeutic Applications.

Protein S-nitrosation (SNO), a posttranslational modification (PTM) of cysteine (Cys) residues elicited by nitric oxide (NO), regulates a wide range of protein functions. As a crucial form of redox-based signaling by NO, SNO contributes significantly to the modulation of physiological functions, and SNO imbalance is closely linked to pathophysiological processes. Site-specific identification of the SNO protein is critical for understanding the underlying molecular mechanisms of protein function regulation. Although careful verification is needed, SNO modification data containing numerous functional proteins are a potential research direction for druggable target identification and drug discovery. Undoubtedly, SNO-related research is meaningful not only for the development of NO donor drugs but also for classic target-based drug design. Herein, we provide a comprehensive summary of SNO, including its origin and transport, identification, function, and potential contribution to drug discovery. Importantly, we propose new views to develop novel therapies based on potential protein SNO-sourced targets.

Design, Synthesis, and Biological Evaluation of Organic Nitrite (NO2-) Donors as Potential Anticerebral Ischemia Agents.

The treatment of ischemic stroke (IS) remains a big challenge in clinics, and it is urgently needed to develop novel, safe, and effective medicines against IS. Here, we report the design, synthesis, and biological evaluation of organic NO2- donors as potential agents for the treatment of IS. The representative compound 4a was able to slowly generate low concentrations of NO2- by reaction with a thiol-containing nucleophile, and the NO2- was selectively converted into NO under ischemic/hypoxia conditions to protect primary rat neurons from oxygen-glucose deprivation and recovery (OGD/R)-induced cytotoxicity by enhancing the Nrf2 signaling and activating the NO/cGMP/PKG pathway. Treatment with 4a at 2 h before or after ischemia mitigated the ischemia/reperfusion-induced brain injury in middle cerebral artery occlusion (MCAO) rats by producing NO and enhancing Nrf2 signaling. Furthermore, 4a significantly promoted endothelial cell proliferation and angiogenesis within the ischemic penumbra. Our findings suggest that this type of NO2- donors, like 4a, may be valuable to fight IS and other ischemic diseases.

A carrier-free anti-inflammatory platinum (II) self-delivered nanoprodrug for enhanced breast cancer therapy.

Cisplatin is one of the most used first-line anticancer drugs for various solid tumor therapies. However, cisplatin-based chemotherapy can induce tumor cells to secrete excessive prostaglandin E2 (PGE2) catalyzed by cyclooxygenase-2 (COX-2), which, in turn, counteracts its chemotherapeutic effect and further accelerates tumor metastasis. Here, we report a carrier-free self-delivered nanoprodrug based on platinum (II) coordination bonding coupled with tolfenamic acid (Tolf) (named Tolfplatin). Tolfplatin can spontaneously assemble into uniformly sized nanoparticles (NPs) with a high drug-loading capacity. Compared with cisplatin, Tolfplatin NPs can facilitate cellular uptake, significantly decrease PGE2 secretion by COX-2 inhibition, which further downregulate tumorous anti-apoptotic and metastasis-associated proteins, thereby efficiently inducing apoptotic cell death and significantly inhibit tumor metastasis in vitro and in vivo. Therefore, as the carrier-free nanoprodrug, Tolfplatin NPs are promising anti-tumoral agents to inhibit tumor proliferation and metastasis by enriching the function and promoting the anti-tumor activity of cisplatin.

Tetrazine-mediated bioorthogonal removal of 3-isocyanopropyl groups enables the controlled release of nitric oxide in vivo.

Bond cleavage bioorthogonal chemistry has been widely employed to restore or activate proteins or prodrugs. Nitric oxide (NO), as a free radical molecule, has joined the clinical arena of cancer therapy, since high levels of NO could produce a cancer cell growth inhibitory effect. However, the spatiotemporal controlled release of NO remains a great challenge, and bioorthogonal chemistry may open a new window. Herein, we described a class of O2-3-isocyanopropyl diazeniumdiolates 3a-f as new bioorthogonal NO precursors, which can be effectively uncaged via tetrazine-mediated bond cleavage reactions to liberate NO and acrolein in living cancer cells, exhibiting potent antiproliferative activity. Furthermore, 3a and tetrazine BTZ were respectively encapsulated into two liposomes. It was found that simultaneous administrations of the two liposomes could specifically release large amounts of NO in the implanted cancer cells in zebrafish, thus generating potent tumor suppression activity in vivo. Our findings indicate that the TZ-labile NO precursors could serve to expand the NO-based smart therapeutics and the scope of bioorthogonal chemistry utility in vivo in the near future.

General Strategy for Integrated Bioorthogonal Prodrugs: Pt(II)-Triggered Depropargylation Enables Controllable Drug Activation In Vivo.

Bioorthogonal decaging reactions for controllable drug activation within complex biological systems are highly desirable yet extremely challenging. Herein, we find a new class of Pt(II)-triggered bioorthogonal cleavage reactions in which Pt(II) but not Pt(IV) complexes effectively trigger the cleavage of O/N-propargyl in a variety of ranges of caged molecules under biocompatible conditions. Based on these findings, we propose a general strategy for integrated bioorthogonal prodrugs and accordingly design a prodrug 16, in which a Pt(IV) moiety is covalently connected with an O2-propargyl diazeniumdiolate moiety. It is found that 16 can be specifically reduced by cytoplasmic reductants in human ovarian cancer cells to liberate cisplatin, which subsequently stimulates the cleavage of O2-propargyl to release large amounts of NO in situ, thus generating synergistic and potent tumor suppression activity in vivo. Therefore, Pt(II)-triggered depropargylation and the integration concept might provide a general strategy for broad applicability of bioorthogonal cleavage chemistry in vivo.

Design, synthesis, and biological evaluation of ligustrazine/resveratrol hybrids as potential anti-ischemic stroke agents.

To search for potent anti-ischemic stroke agents, a series of tetramethylpyrazine (TMP)/resveratrol (RES) hybrids 6a-t were designed and synthesized. These hybrids inhibited adenosine diphosphate (ADP)- or arachidonic acid (AA)-induced platelet aggregation, among them, 6d, 6g-i, 6o and 6q were more active than TMP. The most active compound 6h exhibited more potent anti-platelet aggregation activity than TMP, RES, as well as positive control ticlopidine (Ticlid) and aspirin (ASP). Furthermore, 6h exerted strong antioxidative activity in a dose-dependent manner in rat pheochromocytoma PC12 cells which were treated with hydrogen peroxide (H2O2) or hydroxyl radical (·OH). Importantly, 6h significantly protected primary neuronal cells suffered from oxygen-glucose deprivation/reoxygenation (OGD/R) injury, comparable to an anti-ischemic drug edaravone (Eda). Together, our findings suggest that 6h may be a promising candidate warranting further investigation for the intervention of ischemic stroke.

Correction: O2-(6-Oxocyclohex-1-en-1-yl)methyl diazen-1-ium-1,2-diolates: a new class of nitric oxide donors activatable by GSH/GSTπ with both anti-proliferative and anti-metastatic activities against melanoma.

Correction for 'O2-(6-Oxocyclohex-1-en-1-yl)methyl diazen-1-ium-1,2-diolates: a new class of nitric oxide donors activatable by GSH/GSTπ with both anti-proliferative and anti-metastatic activities against melanoma' by Chengfeng Bai et al., Chem. Commun., 2017, 53, 5059-5062.

Hypoxia alleviation-triggered enhanced photodynamic therapy in combination with IDO inhibitor for preferable cancer therapy.

Photodynamic therapy (PDT) has attracted growing attention in the field of cancer therapy due to its non-invasive intervention and initiation of antitumor immune responses by use of non-toxic photosensitizers (PS) and topical light irradiation. However, inherent hypoxia and immunosuppression mediated by checkpoints in tumors severally impair the efficacy of PDT and PDT-induced immunity. Herein, a multi-functional nanoplatform is rationally constructed by fluorinated polymer nanoparticle saturated with oxygen in advance, which simultaneously encapsulated PS (Ce6) and an indoleamine 2,3-dioxygenase (IDO) inhibitor (NLG919). In particular, the tumor hypoxic microenvironment is obviously relieved and much more reactive oxygen species (ROS) is generated by fluorinated nanoparticle compared with alkylated polymer nanoparticle as a control in vitro and in vivo, this is mainly because the fluorinated polymers are endowed with high oxygen carrying capacity which also contributed to the relief of hypoxia. Meanwhile, compared to PDT alone, the co-encapsulation of IDO inhibitor and PS can further greatly enhance efficacy for inhibiting the growth of primary and abscopal tumors via enhanced T cell infiltration. This study can provide a convenient and practical strategy for enhancing the therapeutic effect of PDT and relieving immune suppression, in turn affording clinical benefits for cancer treatment.

Establishment and Characterization of Pemetrexed-resistant NCI-H460/PMT Cells.

BACKGROUND: Pemetrexed (PMT) is a multitargeted antifolate agent that is used for treating patients with Non-Small Cell Lung Cancer (NSCLC). However, patients have presented clinical responses of drug resistance to PMT. OBJECTIVE: This study aimed to explore the underlying mechanisms of PMT resistance in NSCLC cells. METHODS: PMT-resistant NCI-H460/PMT cells were established by treating with PMT in a concentrationescalation manner. MTT assay and colony formation were performed to detect cell proliferation. Immunofluorescence was used to detect the expression of Ki-67. Transwell assay was performed to measure cell migration ability. qPCR and Western blot were used to detect the mRNA and protein expression levels of indicated genes. Small interfering RNAs (siRNA) were used to knockdown ATP binding cassette subfamily B member 1 (ABCB1) and Thymidylate Synthase (TYMS). RESULTS: This study showed that compared with the parental cells, the NCI-H460/PMT cells displayed weakened proliferation and enhanced cell mobility. In addition, the NCI-H460/PMT cells demonstrated cellular senescence, which might result in PMT resistance. The NCI-H460/PMT cells exhibited cross-resistance to other chemotherapeutics, including fluorouracil, paclitaxel, doxorubicin, etoposide and gemcitabine, possibly because of the upregulated expression of ABCB1. However, the ABCB1 knockdown by siRNA failed to eradicate PMT resistance. Moreover, TYMS, a target of PMT, was obviously upregulated in the resistant cells. The genetic silence of TYMS partially abrogated PMT resistance, suggesting that the overexpression of TYMS was a key resistant mechanism of PMT. CONCLUSION: The overexpression of TYMS was an important resistance mechanism of PMT for KRAS-mutated NCI-H460 cells. Cross-resistance to other chemotherapeutics should be considered in addressing PMT resistance.

Anti-CD24 Antibody-Nitric Oxide Conjugate Selectively and Potently Suppresses Hepatic Carcinoma.

Nitric oxide (NO) has a wide range of potential applications in tumor therapy. However, a targeted delivery system for NO donors has remained elusive, creating a bottleneck that limits its druggability. The antibody-drug conjugate (ADC) is a targeted drug delivery system composed of an antibody linked to an active cytotoxic drug. This design may compensate for the weak targeting ability and various biological functions of the NO donor. In this study, we designed the NO donor HL-2, which had a targeted, cleaved disulfide bond and an attachable maleimide terminal. We conjugated HL-2 with an antibody that targeted CD24 through a thioether bond to generate an ADC-like immunoconjugate, antibody-nitric oxide conjugate (ANC), which we named HN-01. HN-01 showed efficient internalization and significantly increased the release of NO in hepatic carcinoma cells in vitro. HN-01 induced apoptosis of tumor cells and suppressed tumor growth in hepatic carcinoma-bearing nude mice through antibody-dependent co-toxicity; HN-01 also increased NO levels in tumor cells. Collectively, this study expands the concept of ADC and provides an innovative NO donor and ANC to address current challenges in targeted delivery of NO. This new inspiration for an ANC design can also be used in future studies for other molecules with intracellular targets. SIGNIFICANCE: This study is the first to expand the concept of ADC with an antibody-nitric oxide conjugate that suppresses hepatic carcinoma in vitro and in vivo.