A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC_(50) value of 0.25 μM. It was confirmed that APPB inhibits N-glycosylation of β-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC_(50) > 200 μM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, 60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay

Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-based Assays

MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the N-acetylglucosamine (GlcNAc) transformation of lipid I to lipid II during peptidoglycan biosynthesis. Park’s nucleotide has been a convenient biochemical tool to study the function of MraY (phospho-MurNAc-(pentapeptide) translocase) and MurG; however, no fluorescent probe has been developed to differentiate individual processes in the biotransformation of Park’s nucleotide to lipid II via lipid I. Herein, we report a robust assay of MurG using either the membrane fraction of a M. smegmatis strain or a thermostable MraY and MurG of Hydrogenivirga sp. as enzyme sources, along with Park’s nucleotide or Park’s nucleotide-Nε-C6-dansylthiourea and uridine diphosphate (UDP)-GlcN-C6-FITC as acceptor and donor substrates. Identification of both the MraY and MurG products can be performed simultaneously by HPLC in dual UV mode. Conveniently, the generated lipid II fluorescent analogue can also be quantitated via UV–Vis spectrometry without the separation of the unreacted lipid I derivative. The microplate-based assay reported here is amenable to high-throughput MurG screening. A preliminary screening of a collection of small molecules has demonstrated the robustness of the assays and resulted in rediscovery of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC_(50) value of 0.25 μM. It was confirmed that APPB inhibits N-glycosylation of β-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC_(50) > 200 μM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, 60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay

DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC₅₀ value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC₅₀ (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC₅₀: 1.25 μM) inhibits growth of PD002 at 0.0024–0.16 μM in combination with 0.10–2.0 μM CPPB (IC₅₀: 35 μM)

DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC₅₀ value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC₅₀ (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC₅₀: 1.25 μM) inhibits growth of PD002 at 0.0024–0.16 μM in combination with 0.10–2.0 μM CPPB (IC₅₀: 35 μM)