Discovery And Optimization Of Novel Small Molecular Inhibitors Based On New Antifungal Targets Secreted Aspartic Protease And N-myristoyltransferase

Fungal infection includes superficial infection and systemic infection. Superficial infection, a recurrent disease, is spread widely in the world. Systemic infection is often life threatening with a dramatic rise in morbidity and mortality, predominantly in immunocompromised individuals such as HIV patients, transplant recipients, and cancer patients undergoing chemotherapy. Invasive fungal infection caused by Candida albicans leads to a mortality in the range of 20%～40%. However, it was a great challenge to combat with the emergence of fungal strains resistant to antifungal agents in clinical. And the current antifungal therapies also suffered from limited efficacy and narrow spectrum, hepatic and renal toxicity, serious drug-drug interactions, etc. Thus, there is an urgent demand to find new antifungal lead compounds with new mode of actions, higher potency, broader spectrum and lower toxicity.The present dissertation aims to identify novel inhibitors of new potential antifungal targets. Based on the establishment of secreted aspartic protease 2 (Sap2) inhibition assay and virtual screening, a series of novel sap2 inhibitors were identified. The structure activity relationships and bioactivities of the new inhibitors were investigated. In addition, a number of novel N-Myristoyltransferase (NMT) inhibitors were designed and synthesized based on FTR1335.Ⅰ. Discovery and biological evaluation of novel sap2 inhibitors by structure-based virtual screeningIt is a highly challenging task to discover small molecule Sap2 inhibitors because of larger surface area and high flexibility of the active site. Using GOLD as the docking software, forty-five candidates were obtained from the Specs database by virtual screening. Eighteen compounds were supposed to be active with enzyme inhibition rate higher than 40% at the concentration of 100μM. Compounds A8, A12, A39 and A40 showed moderate to good enzyme inhibition activities. In particular, compound A40 exhibited the best activity with IC50 value of 13.78μM. The bind mode of these inhibitors were clarified by molecular docking and molecular dynamics studies. The results revealed that they bound with Sap2 active site through hydrophobic, hydrogen binding and π-π interactions. All the inhibitors were inactive in the in vitro antifungal assay, which was well consistent with the action mode of virulence inhibitors. Notably, compounds A12 and A40 showed good in vivo antifungal activity in the Candida-mediated Caenorhabditis elegans assay, which were considered as good starting points for the development of novel antifungal lead compounds.II. Design, synthesis and biological evaluation of novel thiazolidine-4-one derivatives as Sap2 inhibitorsPreviously, compound A40 was identified as a novel Sap2 inhibitor by virtual screening. According to docking mode and feasibility of synthesis,71 novel thiazolidine-4-one derivatives bearing various substitutions or modified scaffolds were designed and synthesized. Rings A and B are located in hydrophobic pocket S1’ and S1, playing a key role in stabilizing the conformation. Compounds with 4-methyl substitutions on Ring A and B exhibited better activity. The side chain with carboxyl groups on Ring C was an essential component to maintain enzyme inhibition activity, which could enhance the hydrogen bonding interaction with sap2. Compounds D7, D8, D12, D14, D19 and D22 showed excellent inhibition activities with IC50 values less than 5μM. Compounds C2 and D8 exhibited significant in vivo antifungal activities in C. elegans-Candida infection model, which were comparable to fluconazole. In particular, compound D8 showed the best inhibition activity (IC50=0.86μM) and could significantly prolong the survival time in systemic Candida albicans infection mouse model. Moreover, the combination therapy of compound D8 with fluconazole could be an effective strategy to overcome drug resistance.Ⅲ. Design, synthesis and biological evaluation of novel 2,4,6-trisubstituted-1,3,5-triazine derivatives as Sap2 inhibitorsCompound A12 was identified as a Sap2 inhibitor by virtual screening, which showed in vivo antifungal activity in C. elegans-Candida infection model. On the basis of compound A12, thirty-one novel analogs were designed and synthesized. The introduction of various groups on the ring of C4-side chain could have some effect on the inhibition activity; introducing carboxyl groups on C6-side chain was more suitable to enhance enzyme inhibition activity. Compound 130 was the most active one (IC50=9.15μM), which exhibited good in vivo antifungal activity in C. elegans-Candida infection model. Molecular docking was performed to clarify the binding mode between triazine derivatives and sap2, providing reasonable explanation for the SARs.IV. Discovery, design, synthesis and biological evaluation ofnovel pyrazolone derivatives as Sap2 inhibitorsBased on virtual screening of the ChemDiv database, fifty candidates were obtained. Among them, eight compounds showed an enzyme inhibition rate higher than 40% at the concentration of 100μM. Compounds J16, J28, J29 and J41 showed moderate to good enzyme inhibition activities. In particular, compound J29 exhibited the best inhibition activity (IC50=5.04μM). Thirty derivatives based on compound J29 were designed and synthesized to investigate the effects of diverse substitutions. The substitutions on Ring A had various influence on inhibition activity, and the side chain with carboxyl groups on Ring B was necessary to improve the Sap2 inhibition activity. Molecular docking of compounds K13 and K20 demonstrated that new hydrogen bonding interaction with sap2 was formed. The pyrazolone Sap2 inhibitors provided new lead compounds for further structural optimization.V. Design, synthesis and antifungal activities of novel benzothiazole derivatives as N-myristoyltransferase inhibitorsN-myristoyltransferase is a new target enzyme for the development of novel fungicidal drugs, which catalyzes the transfer of the myristoyl group from myristoyl CoA to the N-terminal glycine of a number of eukaryotic cellular and viral proteins. NMT is an essential enzyme in the pathogenicity of C. albicans and C. neoformans. FTR1335 as a NMT inhibitor showed potent antifungal activity, which represented a promising lead for the development of novel antifungal agent.This study highlighted the SARs of FTR1335 by modifying its benzothiazole scaffold and the terminal naphthyl group. Twenty-five novel derivatives were designed and synthesized. Compounds Nl (MIC80=1 g/mL), N2 (MIC8o=1 g/mL) and O1 (MIC80= 0.125 g/mL) showed excellent activities against M. gypseum, which was significantly more potent than fluconazole (MIC=32 g/mL). Compounds L9 and L14 showed broad-spectrum inhibitory activity against most of the tested fungal pathogens, whereas lead compound was only active against C. albicans. Moreover, compound L14 exhibited significant antifungal activity in vivo in the C. elegans-Candida infection model, which is worthy of further optimization.In summary, this thesis focused on the discovery and optimization of novel small molecular inhibitors based on new antifungal targets, secreted aspartic protease and N-myristoyltransferase. Structure-based virtual screening was successfully performed to identify novel Sap2 inhibitors. A total of 131 new derivatives were designed and synthesized to investigate the SARs. Several compounds showed superior enzyme inhibition activities and effective antifungal activities in nematodes or mouse mode. Twenty-five benzothiazole derivatives were designed and synthesized as NMT inhibitors, some of which showed better potency and broader spectrum. This study laid a good foundation on exploring novel antifungal inhibitors with new mechanism of actions.