Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy.

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

Synthesis and Staphylococcus aureus biofilm inhibitory activity of indolenine-substituted pyrazole and pyrimido[1,2-b]indazole derivatives.

Staphylococcus aureus is a highly adaptable opportunistic pathogen that can form biofilms and generate persister cells, leading to life-threatening infections that are difficult to treat with antibiotics alone. Therefore, there is a need for an effective S. aureus biofilm inhibitor to combat this public health threat. In this study, a small library of indolenine-substituted pyrazoles and pyrimido[1,2-b]indazole derivatives were synthesised, of which the hit compound exhibited promising antibiofilm activities against methicillin-susceptible S. aureus (MSSA ATCC 29213) and methicillin-resistant S. aureus (MRSA ATCC 33591) at concentrations significantly lower than the planktonic growth inhibition. The hit compound could prevent biofilm formation and eradicate mature biofilms of MSSA and MRSA, with a minimum biofilm inhibitory concentration (MBIC50) value as low as 1.56 µg/mL and a minimum biofilm eradication concentration (MBEC50) value as low as 6.25 µg/mL. The minimum inhibitory concentration (MIC) values of the hit compound against MSSA and MRSA were 50 µg/mL and 25 µg/mL, respectively, while the minimum bactericidal concentration (MBC) values against MSSA and MRSA were > 100 µg/mL. Preliminary structure-activity relationship analysis reveals that the fused benzene ring and COOH group of the hit compound are crucial for the antibiofilm activity. Additionally, the compound was not cytotoxic to human alveolar A549 cells, thus highlighting its potential as a suitable candidate for further development as a S. aureus biofilm inhibitor.

Improvement of Gene Delivery by Minimal Bacteriophage Particles.

Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed in vitro that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.

A Reverse Structure-based Design of HPV E7 Inhibitor.

BACKGROUND: Human papillomavirus (HPV) is a small, non-enveloped double-stranded circular DNA virus. The high-risk types of HPV are claimed to be responsible for over 99% of cervical cancers. One of the essential HPV oncoproteins, E7, is responsible for escaping from G1/S cell cycle arrest in HPV-infected cells by binding to the retinoblastoma protein (pRb) through its LXCXE binding site. OBJECTIVE: To design a peptide inhibitor targeting HPV E7 through an in silico approach. METHODS: In this study, the LXCXE binding domain of pRb is used as a target to design peptide inhibitors using a reverse structure-based approach. The designed amino acid sequence from the B pocket of pRb, named peptide Y, was further investigated in vitro analysis. The cytotoxicity of the peptide was analysed in two cell lines, namely, CaSki, containing an integrated HPV16 genome, and HaCaT, an immortalized keratinocyte cell. Cell cycle analysis was also carried out in both cell lines treated with peptides. RESULTS: In the in silico approach, a 9-amino acids peptide sequence formed 4 conventional hydrogen bonds with LXCXE motif was selected for in vitro assay. Based on the cytotoxicity analysis, the peptide showed low toxicity in both cell lines, where the cell viability remained over 74% when treated with peptide Y. The peptide also caused an accumulation of cells in G0/G1 (+5.4%) and S phase (+10.2%) and a reduction of cells in the G2/M phase (-14.9%) in the CaSki cells with no significant effect on normal cells, indicating it is a potential HPV inhibitor. CONCLUSION: A peptide inhibitor, peptide Y, that was designed from the LXCXE binding motif in pRb can inhibit HPV E7 by causing a cell accumulation effect in G0/G1, and S phases of the cell cycle in the HPV transformed cell lines. These findings could contribute to HPV E7 peptide inhibitor in the future.

Development of a Novel Cytokine Vehicle Using Filamentous Phage Display for Colorectal Cancer Treatment.

Due to its highly immunogenic nature and the great engineerability, filamentous phage has shown promising antitumor activities in preclinical studies. Previous designs of antitumor phage mainly focused on tumor targeting using a cancer-specific moiety displayed on the minor capsid protein, pIII. In this work, we developed a new therapeutic platform of filamentous phage, in which the major capsid protein pVIII was utilized for displaying an antitumor cytokine. We showcased that a 16.1-kD cytokine GM-CSF could be efficiently presented on the M13 phage particle using the 8 + 8 type display system through a highly tolerable pVIII variant P8(1a). We verified that the GM-CSF phage was a potent activator for STAT5 signaling in murine macrophage. The GM-CSF phage significantly reduced the tumor size by more than 50% as compared to the unmodified phage in a murine colorectal cancer model. Immunological profiling of the tumor-infiltrating leukocytes revealed that an increase of CD4+ lymphocytes in the GM-CSF phage treatment group. Furthermore, the combined therapy of the GM-CSF phage and radiation greatly improved the therapeutic potency with a 100% survival rate and a 25% complete remission rate. We observed that the IFN-γ expression was dramatically up-regulated by the combined therapy in multiple types of tumor-infiltrating immune cells. Overall, we created a novel vehicle for cytokine therapy using the pVIII filamentous phage display. This new platform can be multiplexed with other phage engineering approaches, such as displaying targeting ligands on pIII or encapsulating therapeutic genes inside phage capsids, to create multifunctional nanoparticles for cancer therapy.

Rational drug discovery: Ellagic acid as a potent dual-target inhibitor against hepatitis C virus genotype 3 (HCV G3) NS3 enzymes.

Structure-based virtual screening (SBVS) has served as a popular strategy for rational drug discovery. In this study, we aimed to discover novel benzopyran-based inhibitors that targeted the NS3 enzymes (NS3/4A protease and NS3 helicase) of HCV G3 using a combination of in silico and in vitro approaches. With the aid of SBVS, six novel compounds were discovered to inhibit HCV G3 NS3/4A protease and two phytochemicals (ellagic acid and myricetin) were identified as dual-target inhibitors that inhibited both NS3/4A protease and NS3 helicase in vitro (IC50  = 40.37 ± 5.47 nm and 6.58 ± 0.99 µm, respectively). Inhibitory activities against the replication of HCV G3 replicons were further assessed in a cell-based system with four compounds showed dose-dependent inhibition. Compound P8 was determined to be the most potent compound from the cell-based assay with an EC50 of 19.05 µm. The dual-target inhibitor, ellagic acid, was determined as the second most potent (EC50  = 32.37 µm) and the most selective in its inhibitory activity against the replication of HCV replicons, without severely affecting the viability of the host cells (selectivity index > 6.18).

Discovery of Dengue Virus Inhibitors.

To date, there is still no approved anti-dengue agent to treat dengue infection in the market. Although the only licensed dengue vaccine, Dengvaxia is available, its protective efficacy against serotypes 1 and 2 of dengue virus was reported to be lower than serotypes 3 and 4. Moreover, according to WHO, the risk of being hospitalized and having severe dengue increased in seronegative individuals after they received Dengvaxia vaccination. Nevertheless, various studies had been carried out in search of dengue virus inhibitors. These studies focused on the structural (C, prM, E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) of dengue virus as well as host factors as drug targets. Hence, this article provides an overall up-to-date review of the discovery of dengue virus inhibitors that are only targeting the structural and non-structural viral proteins as drug targets.

Sofosbuvir as treatment against dengue?

Dengvaxia® (CTD-TDV), the only licensed tetravalent dengue vaccine by Sanofi Pasteur, was made available since 2015. However, administration of CTD-TDV, in general, has not received the prequalification recommendation from the World Health Organization. Having a universal antidengue agent for treatment will therefore beneficial. Accordingly, the development of nucleoside inhibitors specific to dengue viral polymerase that perturb dengue infection has been studied by many. Alternatively, we have used a marketed anti-HCV prodrug sofosbuvir to study its in silico and in vitro effects against dengue. As a result, the active metabolite of sofosbuvir (GS-461203) was predicted to bind to the catalytic motif (Gly-Asp-Asp) of dengue viral polymerase with binding affinity of -6.9 kcal/mol. Furthermore, sofosbuvir demonstrated excellent in vitro viral inhibition with an EC90 of 0.4 µm. In addition, this study demonstrated the requirement of specific liver enzymes to activate the prodrug into GS-461203 to exert its antidengue potential. All in all, sofosbuvir should be subjected to in-depth studies to provide information of its efficacy toward dengue and its lead potential as DENV polymerase inhibitor in human subjects. In conclusion, we have expended the potential of the clinically available drug sofosbuvir as treatment for dengue.