Therapeutic Efficacy In Mouse Models Research Articles

Discovery of a Bromodomain and Extra Terminal Domain (BET) Inhibitor with the Selectivity for the Second Bromodomain (BD2) and the Capacity for the Treatment of Inflammatory Diseases.

Selective inhibitors targeting the first bromodomain (BD1) or the second bromodomain (BD2) of the bromodomain and extra terminal domain (BET) proteins have triggered extensive research to produce more specific agents. Herein, we described our efforts to design and synthesize a series of selective BET BD2 inhibitors with novel structures. Among them, compound 45 showed single-digit nanomolar potency against BRD4 BD2 (IC50: 1.6 nM) and a 328-fold selectivity for BRD4 BD2 over BRD4 BD1 (IC50: 524 nM). Besides, 45 possessed potent effects on regulating the differentiation of Th17 cells and reducing the levels of Th17-related cytokines by affecting the activation of STAT3 and NF-κB. Further studies demonstrated that 45 had significant therapeutic efficacy in mouse models of imiquimod (IMQ)-induced psoriasis and dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD). This work provides a strong foundation for the development of selective BET BD2 inhibitors and the therapeutic strategy for psoriasis and IBD.

PTEN potentiation of oncolytic HSV therapy for glioblastoma

PTEN potentiation of oncolytic HSV therapy for glioblastoma

Characterization of an allosteric inhibitor of fungal-specific C-24 sterol methyltransferase to treat Candida albicans infections

Filamentation is an important virulence factor of the pathogenic fungus Candida albicans. The abolition of Candida albicans hyphal formation by disrupting sterol synthesis is an important concept for the development of antifungal drugs with high safety. Here, we conduct a high-throughput screen using a C. albicans strain expressing green fluorescent protein-labeled Dpp3 to identify anti-hypha agents by interfering with ergosterol synthesis. The antipyrine derivative H55 is characterized to have minimal cytotoxicity and potent inhibition of C. albicans hyphal formation in multiple cultural conditions. H55 monotherapy exhibits therapeutic efficacy in mouse models of azole-resistant candidiasis. H55 treatment increases the accumulation of zymosterol, the substrate of C-24 sterol methyltransferase (Erg6). The results of enzyme assays, photoaffinity labeling, molecular simulation, mutagenesis, and cellular thermal shift assays support H55 as an allosteric inhibitor of Erg6. Collectively, H55, an inhibitor of the fungal-specific enzyme Erg6, holds potential to treat C. albicans infections.

Curcumin Diethyl γ-Aminobutyrate, a Prodrug of Curcumin, for Enhanced Treatment of Inflammatory Pain.

Curcumin is a naturally occurring polyphenol compound with potential analgesic effects. It has been shown to improve pain-like behaviors in numerous models of pain. Despite its potential, curcumin exhibits poor physicochemical and pharmacokinetic properties, which hinder its oral therapeutic efficacy. Curcumin diethyl γ-aminobutyrate (CUR-2GE), a carbamate prodrug of curcumin, was designed to overcome these limitations and demonstrated greater anti-neuroinflammatory effects compared to curcumin in vitro. Thus, this study evaluated the effect of CUR-2GE and its parent compound on pain-like behaviors in carrageenan- and LPS-induced mouse models. The possible side effects of CUR-2GE were also assessed by exploring its effects on motor coordination and spontaneous locomotor activity after acute and chronic treatments. The results showed that CUR-2GE improved mechanical and thermal hyperalgesia and locomotor activity to a greater extent than curcumin in carrageenan-induced mice. These results are in line with the ability of CUR-2GE to suppress peripheral inflammation in the paw tissue of carrageenan-induced mice, indicated by a significant decrease in TNF-α and IL-6 expression levels. Similarly, in LPS-induced mice, CUR-2GE improved sickness and pain-like behaviors (exploratory behaviors and long-term locomotor activity) to a greater extent than curcumin. Furthermore, CUR-2GE significantly reduced the level of proinflammatory cytokines in both the plasma and spinal cord tissue of LPS-induced mice, exhibiting significantly higher inhibition than curcumin. Moreover, the motor coordination, and locomotive behaviors of mice were not affected by both acute and chronic administration of CUR-2GE, indicating no potential CNS side effects. Thus, CUR-2GE demonstrated enhanced therapeutic efficacy in mouse models of inflammatory pain without any possible CNS side effects, suggesting its potential to be developed as an analgesic agent against inflammatory pain.

Single-Domain Antibodies as Therapeutics for Respiratory RNA Virus Infections.

Over the years, infectious diseases with high morbidity and mortality disrupted human healthcare systems and devastated economies globally. Respiratory viruses, especially emerging or re-emerging RNA viruses, including influenza and human coronavirus, are the main pathogens of acute respiratory diseases that cause epidemics or even global pandemics. Importantly, due to the rapid mutation of viruses, there are few effective drugs and vaccines for the treatment and prevention of these RNA virus infections. Of note, a class of antibodies derived from camelid and shark, named nanobody or single-domain antibody (sdAb), was characterized by smaller size, lower production costs, more accessible binding epitopes, and inhalable properties, which have advantages in the treatment of respiratory diseases compared to conventional antibodies. Currently, a number of sdAbs have been developed against various respiratory RNA viruses and demonstrated potent therapeutic efficacy in mouse models. Here, we review the current status of the development of antiviral sdAb and discuss their potential as therapeutics for respiratory RNA viral diseases.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation.

Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.

A deimmunized and pharmacologically optimized Toll-like receptor 5 agonist for therapeutic applications

The Toll-like receptor 5 (TLR5) agonist entolimod, a derivative of Salmonella flagellin, has therapeutic potential for several indications including radioprotection and cancer immunotherapy. However, in Phase 1 human studies, entolimod induced a rapid neutralizing immune response, presumably due to immune memory from prior exposure to flagellated enterobacteria. To enable multi-dose applications, we used structure-guided reengineering to develop a next-generation, substantially deimmunized entolimod variant, GP532. GP532 induces TLR5-dependent NF-κB activation like entolimod but is smaller and has mutations eliminating an inflammasome-activating domain and key B- and T-cell epitopes. GP532 is resistant to human entolimod-neutralizing antibodies and shows reduced de novo immunogenicity. GP532 also has improved bioavailability, a stronger effect on key cytokine biomarkers, and a longer-lasting effect on NF-κB. Like entolimod, GP532 demonstrated potent prophylactic and therapeutic efficacy in mouse models of radiation-induced death and tissue damage. These results establish GP532 as an optimized TLR5 agonist suitable for multi-dose therapies and for patients with high titers of preexisting flagellin-neutralizing antibodies.

Fibroblast growth factor signalling in osteoarthritis and cartilage repair.

Regulated fibroblast growth factor (FGF) signalling is a prerequisite for the correct development and homeostasis of articular cartilage, as evidenced by the fact that aberrant FGF signalling contributes to the maldevelopment of joints and to the onset and progression of osteoarthritis. Of the four FGF receptors (FGFRs 1-4), FGFR1 and FGFR3 are strongly implicated in osteoarthritis, and FGFR1 antagonists, as well as agonists of FGFR3, have shown therapeutic efficacy in mouse models of spontaneous and surgically induced osteoarthritis. FGF18, a high affinity ligand for FGFR3, is the only FGF-based drug currently in clinical trials for osteoarthritis. This Review covers the latest advances in our understanding of the molecular mechanisms that regulate FGF signalling during normal joint development and in the pathogenesis of osteoarthritis. Strategies for FGF signalling-based treatment of osteoarthritis and for cartilage repair in animal models and clinical trials are also introduced. An improved understanding of FGF signalling from a structural biology perspective, and of its roles in skeletal development and diseases, could unlock new avenues for discovery of modulators of FGF signalling that can slow or stop the progression of osteoarthritis.

Efficacy of the Combination of Metformin and CTLA4Ig in the (NZB × NZW)F1 Mouse Model of Lupus Nephritis.

CTLA4Ig, a reagent that inhibits CD28 signaling, has shown therapeutic efficacy in mouse models of lupus nephritis (LN) when combined with several other biologics or standard of care drugs. Unfortunately, clinical trials treating LN patients with CTLA4Ig (abatacept) have not met endpoints. Metformin, a drug used to control hyperglycemia that inhibits mitochondrial metabolism, lowered the effective dose of glucocorticoids and prevented major flares when added on to the standard of care treatment of lupus patients with low disease activity. Metformin combined with inhibition of glycolysis by 2-deoxyglucose showed therapeutic efficacy in multiple mouse models of LN. Because CD28 signaling triggers glucose metabolism in T cells, we hypothesized that combining CTLA4Ig treatment with metformin would have the same effect. In this study, we showed that the combination of metformin and CTLA4Ig decreased the development of LN in (NZB × NZW)F1 mice treated at the early stage of disease. This preventive effect was associated with a decreased expansion of CD4+ T cell effector subsets. However, contrary to the combination with 2-deoxyglucose, metformin combined with CTLA4Ig did not alter autoantibody production, suggesting different mechanisms of symptom mitigation. Overall, this study shows therapeutic efficacy of the combination of metformin and CTLA4Ig, two drugs with established safety records, in a preclinical mouse model of LN.

PAD enzymes in rheumatoid arthritis: pathogenic effectors and autoimmune targets

Peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) owing to their ability to generate citrullinated proteins - the hallmark autoantigens of RA. Of the five PAD enzyme isoforms, PAD2 and PAD4 are the most strongly implicated in RA at both genetic and cellular levels, and PAD inhibitors have shown therapeutic efficacy in mouse models of inflammatory arthritis. PAD2 and PAD4 are additionally targeted by autoantibodies in distinct clinical subsets of patients with RA, suggesting anti-PAD antibodies as possible biomarkers for RA diagnosis and prognosis. This Review weighs the evidence that supports a pathogenic role for PAD enzymes in RA as both promoters and targets of the autoimmune response, as well as discussing the mechanistic and therapeutic implications of these findings in the wider context of RA pathogenesis. Understanding the origin and consequences of dysregulated PAD enzyme activity and immune responses against PAD enzymes will be important to fully comprehend the pathogenic mechanisms involved in this disease and for the development of novel strategies to treat and prevent RA.

Enzyme-responsive multifunctional peptide coating of gold nanorods improves tumor targeting and photothermal therapy efficacy

It is well known that stealth coating effectively extends the circulation lifetime of nanomaterials in blood, which favors systemic delivery but also limits their cellular internalization and in turn prevents efficient tumor-targeting and accumulation. In this study, we address this dilemma by developing an enzyme-responsive zwitterionic stealth peptide coating capable of responding to matrix metalloproteinase-9 (MMP-9) which is overexpressed in tumor microenvironment. The peptide consists of a cell-penetrating Tat sequence, an MMP-9 cleavable sequence, and a zwitterionic antifouling sequence. Using this coating to protect photothermal gold nanorods (AuNRs), we found that responsive AuNRs showed both satisfactory systemic circulation lifetime and significantly enhanced cellular uptake in tumors, resulting in clearly improved photothermal therapeutic efficacy in mouse models. These results suggest that multifunctional peptide coated AuNRs sensitive to MMP-9 are promising nanomaterials, conferring both extended systemic circulation and enhanced tumor tissue accumulation, for more specific and efficient tumor therapy. Statement of significanceIt is well known that stealth coating effectively extends the circulation lifetime of nanomaterials in blood, which favors systemic delivery but also limits their cellular internalization and in turn prevents efficient tumor-targeting and accumulation. In this study, we address this dilemma by developing an enzyme-responsive zwitterionic stealth peptide coating capable of responding to matrix metalloproteinase-9 (MMP-9) which is overexpressed in tumor microenvironment. The peptide consists of a cell-penetrating Tat sequence, an MMP-9 cleavable sequence, and a zwitterionic antifouling sequence. Using this coating to protect photothermal gold nanorods (AuNRs), we found that responsive AuNRs showed both satisfactory systemic circulation lifetime and significantly enhanced cellular uptake in tumors, resulting in clearly improved photothermal therapeutic efficacy in mouse models.

Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) critically contribute to the efficacy of anti-tumor therapeutic antibodies. We report here an unexpected finding that macrophages after ADCP inhibit NK cell-mediated ADCC and Tcell-mediated cytotoxicity in breast cancers and lymphomas. Mechanistically, AIM2 is recruited to the phagosomes by FcγR signaling following ADCP and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosomal membrane, which subsequently upregulates PD-L1 and IDO and causes immunosuppression. Combined treatment with anti-HER2 antibody and inhibitors of PD-L1 and IDO enhances anti-tumorimmunity and anti-HER2 therapeutic efficacy in mouse models. Furthermore, neoadjuvant trastuzumab therapy significantly upregulates PD-L1 and IDO in the tumor-associated macrophages (TAMs) of HER2+ breast cancer patients, correlating with poor trastuzumab response. Collectively, our findings unveil a deleterious role of ADCP macrophages in cancer immunosuppression and suggest that therapeuticantibody plus immune checkpoint blockade may provide synergistic effects in cancer treatment.

AAV9-mediated telomerase activation does not accelerate tumorigenesis in the context of oncogenic K-Ras-induced lung cancer.

Short and dysfunctional telomeres are sufficient to induce a persistent DNA damage response at chromosome ends, which leads to the induction of senescence and/or apoptosis and to various age-related conditions, including a group of diseases known as “telomere syndromes”, which are provoked by extremely short telomeres owing to germline mutations in telomere genes. This opens the possibility of using telomerase activation as a potential therapeutic strategy to rescue short telomeres both in telomere syndromes and in age-related diseases, in this manner maintaining tissue homeostasis and ameliorating these diseases. In the past, we generated adeno-associated viral vectors carrying the telomerase gene (AAV9-Tert) and shown their therapeutic efficacy in mouse models of cardiac infarct, aplastic anemia, and pulmonary fibrosis. Although we did not observe increased cancer incidence as a consequence of Tert overexpression in any of those models, here we set to test the safety of AAV9-mediated Tert overexpression in the context of a cancer prone mouse model, owing to expression of oncogenic K-ras. As control, we also treated mice with AAV9 vectors carrying a catalytically inactive form of Tert, known to inhibit endogenous telomerase activity. We found that overexpression of Tert does not accelerate the onset or progression of lung carcinomas, even when in the setting of a p53-null background. These findings indicate that telomerase activation by using AAV9-mediated Tert gene therapy has no detectable cancer-prone effects in the context of oncogene-induced mouse tumors.

PH-sensitive zwitterionic coating of gold nanocages improves tumor targeting and photothermal treatment efficacy

Stealth coating materials effectively extend a nanoparticle’s systemic circulation lifetime yet limit its cellular internalization, which promotes and prevents tumor targeting, respectively. Here, this contradiction was resolved by using an acutely pH-sensitive zwitterionic stealth ligand capable of responding to small differences in extracellular pH between blood and tumors. Using a photothermal gold nanocage (AuNC) as a model nanotherapeutic, we found that stealth-AuNC nanoparticles showed both significantly enhanced cell uptake efficiency in acidic tumors and a markedly extended systemic circulation lifetime compared to its unaltered analogue. As a result, stealth-AuNC nanoparticles administered intravenously showed significantly enhanced accumulation within the tumor, leading to significantly improved photothermal therapeutic efficacy in mouse models. These results suggests that pH-sensitive zwitterionic ligands with sufficient sensitivity for responding to small differences in extracellular pH between blood and tumors are ideal stealth materials for simultaneously conferring both extended systemic circulation and enhanced cellular internalization, reducing the need for active targeting moieties.

Transglutaminase 2 Inhibition Reverses Mesenchymal Transdifferentiation of Glioma Stem Cells by Regulating C/EBPβ Signaling.

Necrosis is a hallmark of glioblastoma (GBM) and is responsible for poor prognosis and resistance to conventional therapies. However, the molecular mechanisms underlying necrotic microenvironment-induced malignancy of GBM have not been elucidated. Here, we report that transglutaminase 2 (TGM2) is upregulated in the perinecrotic region of GBM and triggered mesenchymal (MES) transdifferentiation of glioma stem cells (GSC) by regulating master transcription factors (TF), such as C/EBPβ, TAZ, and STAT3. TGM2 expression was induced by macrophages/microglia-derived cytokines via NF-κB activation and further degraded DNA damage-inducible transcript 3 (GADD153) to induce C/EBPβ expression, resulting in expression of the MES transcriptome. Downregulation of TGM2 decreased sphere-forming ability, tumor size, and radioresistance and survival in a xenograft mouse model through a loss of the MES signature. A TGM2-specific inhibitor GK921 blocked MES transdifferentiation and showed significant therapeutic efficacy in mouse models of GSC. Moreover, TGM2 expression was significantly increased in recurrent MES patients and inversely correlated with patient prognosis. Collectively, our results indicate that TGM2 is a key molecular switch of necrosis-induced MES transdifferentiation and an important therapeutic target for MES GBM. Cancer Res; 77(18); 4973-84. ©2017 AACR.

High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activators.

We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

Therapeutic Efficacy of a Family of pHLIP-MMAF Conjugates in Cancer Cells and Mouse Models.

The targeting of therapeutics specifically to diseased tissue is crucial for the development of successful cancer treatments. The approach here is based on the pH(low) insertion peptide (pHLIP) for the delivery of a potent mitotic inhibitor monomethyl auristatin F (MMAF). We investigated six pHLIP variants conjugated to MMAF to compare their efficacy in vitro against cultured cancer cells. While all pHLIP-MMAF conjugates exhibit potent pH- and concentration-dependent killing, their cytotoxicity profiles are remarkably different. We also show that the lead conjugate exhibits significant therapeutic efficacy in mouse models without overt toxicities. This study confirms pHLIP-monomethyl auristatin conjugates as possible new therapeutic options for cancer treatment and supports their further development.

Nanoparticle delivery of chemotherapy combination regimen improves the therapeutic efficacy in mouse models of lung cancer

The combination chemotherapy regimen of cisplatin (CP) and docetaxel (DTX) is effective against a variety of cancers. However, combination therapies present unique challenges that can complicate clinical application, such as increases in toxicity and imprecise exposure of tumors to specific drug ratios that can produce treatment resistance. Drug co-encapsulation within a single nanoparticle (NP) formulation can overcome these challenges and further improve combinations' therapeutic index. In this report, we employ a CP prodrug (CPP) strategy to formulate poly(lactic-co-glycolic acid)–poly(ethylene glycol) (PLGA–PEG) NPs carrying both CPP and DTX. The dually loaded NPs display differences in drug release kinetics and in vitro cytotoxicity based on the structure of the chosen CPP. Furthermore, NPs containing both drugs showed a significant improvement in treatment efficacy versus the free drug combination in vivo.