Targeted inactivation of Salmonella Agona metabolic genes by group II introns and in vivo assessment of pathogenicity and anti-tumour activity in mouse model

The fight against cancer has been a never-ending battle. Limitations of conventional therapies include lack of selectivity, poor penetration and highly toxic to the host. Using genetically modified bacteria as a tumour therapy agent has gained the interest of scientist from the past few decades. Low virulence and highly tolerability of Salmonella spp. in animals and humans make it as the most studied pathogen with regards to anti-tumour therapy. The present study aims to construct a genetically modified S. Agona auxotroph as an anti-tumour agent. LeuB and ArgD metabolic genes in ΔSopBΔSopD double knockout S. Agona were successfully knocked out using a Targetron gene knockout system. The knockout was confirmed by colony PCR and the strains were characterized in vitro and in vivo. The knockout of metabolic genes causes significant growth defect in M9 minimal media. Quadruple knockout ΔSopBΔSopDΔLeuBΔArgD (BDLA) exhibited lowest virulence among all of the strains in all parameters including bacterial load, immunity profile and histopathology studies. In vivo anti-tumour study on colorectal tumour bearing-BALB/c mice revealed that all strains of S. Agona were able to suppress the growth of the large solid tumour as compared with negative control and ΔLeuBΔArgD (LA) and BDLA auxotroph showed better efficacy. Interestingly, higher level of tumour growth suppression was noticed in large tumour. However, multiple administration of bacteria dosage did not increase the tumour suppression efficacy. In this study, the virulence of BDLA knockout strain was slightly reduced and tumour growth suppression efficacy was successfully enhanced, which provide a valuable starting point for the development of S. Agona as anti-tumour agent

Targeted inactivation of Salmonella Agona metabolic genes by group II introns and in vivo assessment of pathogenicity and anti-tumour activity in mouse model

The fight against cancer has been a never-ending battle. Limitations of conventional therapies include lack of selectivity, poor penetration and highly toxic to the host. Using genetically modified bacteria as a tumour therapy agent has gained the interest of scientist from the past few decades. Low virulence and highly tolerability of Salmonella spp. in animals and humans make it as the most studied pathogen with regards to anti-tumour therapy. The present study aims to construct a genetically modified S. Agona auxotroph as an anti-tumour agent. LeuB and ArgD metabolic genes in ΔSopBΔSopD double knockout S. Agona were successfully knocked out using a Targetron gene knockout system. The knockout was confirmed by colony PCR and the strains were characterized in vitro and in vivo. The knockout of metabolic genes causes significant growth defect in M9 minimal media. Quadruple knockout ΔSopBΔSopDΔLeuBΔArgD (BDLA) exhibited lowest virulence among all of the strains in all parameters including bacterial load, immunity profile and histopathology studies. In vivo anti-tumour study on colorectal tumour bearing-BALB/c mice revealed that all strains of S. Agona were able to suppress the growth of the large solid tumour as compared with negative control and ΔLeuBΔArgD (LA) and BDLA auxotroph showed better efficacy. Interestingly, higher level of tumour growth suppression was noticed in large tumour. However, multiple administration of bacteria dosage did not increase the tumour suppression efficacy. In this study, the virulence of BDLA knockout strain was slightly reduced and tumour growth suppression efficacy was successfully enhanced, which provide a valuable starting point for the development of S. Agona as anti-tumour agent

Comparative analysis of pathogenicity profiles and antitumour effects of wild and mutant strains of Salmonella enterica serovar Agona

The fight against cancer has been a never ending battle. Cancer remains a major threat to human life. Limitations of conventional therapies included lack of selectivity, poor penetration and highly toxic to the host. One of the innovative approaches which have gained the interest of scientists from past few decades is the use of live, genetically modified bacteria as tumour therapy agent. Engineered bacteria possess unique features to overcome the limitations of conventional therapies. Low virulence and highly tolerability of Salmonella spp. in animals and humans make it as a most studied pathogen in regards to antitumour therapy. Genetically modified S. Typhimurium has been constructed as direct tumouricidal agent or as drug delivery vector in many researches. Main objective of this study is to construct genetically modified S. Agona as antitumour agent. A powerful genetic manipulation tool is needed in order to meet the requirements as tumouricidal agent in experimental and clinical research. Group II intron mutagenesis technology was exploited in current study to inactivate the metabolic genes in S. Agona. Group II intron technology has been shown to be able to insert at desired DNA at high frequency and specificity. In this study, LeuB and ArgD metabolic genes in S. Agona were successfully knockout at frequency of 15% and 3% respectively. Non-reverting and high stability of intron insertion was proven with a stability passage assay of 30 days culture. The constructed knockout S. Agona has become auxotrophic for leucine and arginine. Inactivation of LeuB and ArgD genes leads to a significant growth defect in M9 minimal media. Salmonella is a natural pathogen of mice, thus, mouse model was used to evaluate the potential pathogenicity and antitumour activity of engineered S. Agona in present work. Quadruple knockout BDLA exhibited highest safety among all of the strains in all tested parameters including bacterial colony forming units, immunity profile and histopathology studies. Tumour growth inhibition study was divided into two groups, which are small tumour model with size approximately 250 mm3 and large tumour model with size approximately 450 mm3 in comparison with each other.Results have shown that all of the stains are able to delay the growth of the small and large solid tumour as compare to the negative control, with better efficacy shown by auxotrophic knockout strain LA and BDLA. Interestingly, tumour growth inhibition noticed on small tumour is not as effective as seen on large tumour, might be due to the better hypoxia or nutrient conditions available in microenvironment of big tumour. Furthermore, findings from this study showed that the treated groups with repeated treatment did not show any significant improvement in tumour growth delay, in both big and small solid tumour models. Overall, the virulence of BDLA knockout strain was reduced and antitumour effect was successfully enhanced. The results obtained from current work suggest a great potential of auxotrophic quadruple knockout S. Agona as antitumour agent

Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase

10.1021/acsmedchemlett.9b00619ACS Medicinal Chemistry Letter

Amidoxime prodrugs convert to potent cell-active multimodal inhibitors of the dengue virus protease

https://doi.org/10.1016/j.ejmech.2021.11369

Cell-active carbazole derivatives as inhibitors of the zika virus protease

Zika virus (ZIKV) infection recently resulted in an international health emergency the Americas in and despite its high profile there is currently no approved treatment for ZIKV infection with millions of people being at risk. ZIKV is a member of Flaviviridae family which includes prominent members such as dengue virus (DENV) and West Nile virus (WNV). One of the best validated targets for developing anti-flaviviral treatment for DENV and WNV infection is the NS2B/NS3 protease. However the inhibitors reported to date have shown limited promise for further clinical development largely due to poor cellular activity. Prompted by the conserved nature of the viral NS2B/NS3 protease across flaviviruses, we envisaged that small molecule inhibitors of the ZIKVpro may be developed by applying rational design on previously reported scaffolds with demonstrated activity against other flaviviral proteases. Starting with an earlier WNVpro hit we performed a scaffold hopping exercise and discovered that certain carbazole derivatives bearing amidine groups possessed submicromolar potency and significant cellular activity against ZIKV. We successfully addressed various issues with the synthesis of novel N-substituted carbazole-based amidines thus permitting a targeted SAR campaign. The in vitro biochemical and cell-based inhibitory profiles exhibited by the lead molecule described in this work (ZIKVpro IC50 0.52 μM, EC50 1.25 μM), is among the best reported to date. Furthermore, these molecules possess capacity for further optimization of pharmacokinetics and may evolve to broad spectrum flaviviral protease inhibitors.National Research Foundation (NRF)The research was supported by National Medical Research Council grant NMRC/CBRG/0103/2016 (PI), National Research Foundation grant NRF2016NRF-CRP001-063 (Co-PI) to SGV

Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-a and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells

10.1021/acsinfecdis.8b0037