The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Synthesis of hydrophobic N'-mono and N',N"-double alkylated eremomycins inhibiting the transglycosylation stage of bacterial cell wall biosynthesis.

A series of hydrophobic N'-mono and N',N"-double alkylated derivatives of the glycopeptide antibiotic eremomycin were synthesized by reductive alkylation after preliminary protection of the N-terminal amino group of the peptide backbone. The investigation of the antibacterial activity in vitro showed that N'-C10H21- and N'-p-(p-chlorophenyl)benzyl derivatives of eremomycin are the most active against vancomycin-resistant enterococci among the compounds obtained though they are less effective than the corresponding lipophilic derivatives of vancomycin. The introduction of two hydrophobic substituents led to a decrease in activity against both susceptible and resistant bacteria. The biochemical evaluation of the mode of action revealed that in addition to binding to D-Ala-D-Ala these compounds also have an alternative mechanism of action that does not require substrate binding.

Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

In situ assay for identifying inhibitors of bacterial transglycosylase.

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.

Differential antibacterial activity of moenomycin analogues on gram-positive bacteria.

The moenomycin trisaccharide degradation product and synthetic disaccharide analogues based on the disaccharide core were bactericidal to gram-positive bacteria, inhibited lipid II polymerization, and inhibited cell wall synthesis in Enterococcus faecalis. Truncating moenomycin to the trisaccharide, and building upon the core disaccharide have both led to molecules possessing properties not shared with their respective parent structures.

Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase.

Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding.

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

Synthesis and biological evaluation of analogues of bacterial lipid I.

Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG.

Targeting cell wall synthesis and assembly in microbes: similarities and contrasts between bacteria and fungi.

icrobial cells possess a form of exoskeleton called the cell wall that protects the organism from osmotic pressure and environmental insults. Synthesis of the various building blocks that make up the cell wall occurs in the cytoplasm, and thus microbial cells face specific biochemical and biophysical problems related to the polymerization, transport, and assembly of building blocks into the final wall structure at an extra-cellular site. Cell walls must also be metabolically and structurally pliable in order to allow for processes such as repair, secretion, DNA exchange, and cell division. In some cases, bacteria and fungi use similar mechanisms, to accomplish synthesis and assembly, while in other cases each used divergent strategies to accomplish specific functions. This review will summarize recent advances in our understanding of fungal and bacterial cell wall synthesis and assembly. We will compare specific pathways used by both fungi and bacteria, paying particular attention to identifying those areas where what is known in one system may point to approaches to solving unanswered questions in the other. The structure, chemical properties, and mechanism of action of select natural and synthetic products which inhibit synthesis or assembly of cells walls will be discussed in terms of similarities in the structures, and/or steps in the synthetic process targeted. In addition, new targets in the pathways will be presented along with recent approaches to the discovery and design of novel inhibitors.

Attenuated bacteria as a DNA delivery vehicle for DNA-mediated immunization.

Previously, Shigella carrier 15D was shown to deliver a mammalian DNA expression plasmid to cultured cells with subsequent production of the plasmid-encoded foreign protein. In this study, we report in vivo delivery of a DNA expression plasmid to mucosal tissue results in the stimulation of immune responses against the plasmid-encoded foreign antigen. Splenocytes from mice receiving two intranasal inoculations of 15D carrying pCMV beta showed proliferative responses to the plasmid-encoded Escherichia coli beta-galactosidase. In addition, antibody specific for beta-galactosidase was detected in pooled sera collected from 15D (pCMV beta) infected mice.

Interaction of Salmonella typhi strains with cultured human monocyte-derived macrophages.

Human monocyte-derived macrophages (MDM) provided this laboratory with a tool to develop a primary-cell assay for evaluating the relative virulence of newly constructed Salmonella typhi carrier strains. In this study, the interaction with and survival within MDM were compared for delta aroA143-attenuated strains, wild-type virulent strains, and the current oral-vaccine strain, Ty21a.

Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization.

Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

Effects of FP2 and a mercury resistance plasmid from Pseudomonas aeruginosa PA103 on exoenzyme production.

Plasmids encoding mercury resistance carried by Pseudomonas aeruginosa PAO1161 and PA103 were found to be involved in regulating the secretion of protease, phospholipase C, and alkaline phosphatase. Previously, mutations in Pseudomonas strains that caused pleiotropic effects on the production of extracellular enzymes were mapped to the bacterial chromosome. We show that pleiotropic changes in extracellular enzyme production can also be regulated by plasmids. In this study, the effects on secretion of exoenzymes by two mercury resistance plasmids, FP2 from PAO1161 and pRLW103 from PA103, were assayed in P. aeruginosa PAO1 and PAO18. The introduction of either plasmid into PAO1 resulted in a significant decrease in exoprotease production. Additionally, pRLW103 significantly increased the production of alkaline phosphatase by both strains. Phospholipase C was produced only in strain PAO18 containing the pRLW103 plasmid. FP2 had no effect on alkaline phosphatase or phospholipase C production in either strain and was found to decrease exoprotease secretion only in strain PAO1. The results indicate the P. aeruginosa mercury resistance plasmids vary in their ability to modify exoenzyme expression, and this ability is influenced by the host strain.

Detection of restriction fragment length polymorphisms in clinical isolates and serially passaged Pseudomonas aeruginosa strains.

An 800-base-pair HindIII-PstI fragment that flanks a hot spot for Tn7 insertion was isolated from the chromosome of Pseudomonas aeruginosa and cloned into pUC12. The fragment was used to probe XhoI digests of genomic DNA from 18 P. aeruginosa isolates collected from sputum samples of seven cystic fibrosis patients. Only two XhoI restriction fragment length polymorphisms (RFLPs), of 3.7 and 7.7 kilobases (kb), were detected. Isolate WSU3531-1 (3.7-kb XhoI fragment) and WSU3860 (7.7-kb XhoI fragment), while isolated from the same patient, showed different RFLPs. Serial passages of isolate WSU3531-1 demonstrated that this strain was phenotypically stable. In contrast, colony and pigment variants were readily isolated at a frequency of 1% from serial passages of isolate WSU3860. When XhoI-digested genomic DNA from phenotypic variants of serially passaged WSU3860 were probed with the 800-base-pair HindIII-PstI fragment, the probe hybridized to a 10.4-kb XhoI fragment from three isolates. Restriction analysis of the genomic DNA digested with a variety of restriction enzymes showed that a 2.7-kb insertion occurred in the same region for all three isolates. There appeared to be no correlation between changes in the RFLP and changes in colony morphology.