Acute myeloid leukemia (AML) is an aggressive malignancy with only a handful of therapeutic options. About 30% of AML patients harbor mutated FLT3 kinase, and thus, this cancer-driver has become a hotly pursued AML target. Herein we report a new class of FLT3 inhibitors, which potently inhibit the proliferation of acute myeloid leukemia (AML) cells at nanomolar concentrations.Keywords: acute myeloid leukemia (AML); azo; click-it/staple-it; FMS-like tyrosine kinase 3 (FLT3); inhibitor;

Cyclic dinucleotide-based second messengers, including c-di-GMP, c-di-AMP and cGAMP, are universal signaling molecules widely used by prokaryotes and eukaryotes. C-di-GMP and c-di-AMP play key roles in bacterial survival. Consequently, metabolism proteins that modulate cyclic dinucleotide concentrations, such as cyclic dinucleotide phosphodiesterases (PDE), are potential drug targets; thus, probes that report PDE activity could have great utility in high throughput screening for PDE inhibitors. However, there is a paucity of luminescent-based probes for monitoring cyclic dinucleotide PDE activity. In this study, we synthesized various fluorescent nucleobase (ethenoA and 2-AP) analogs of cyclic and linear dinucleotides. These analogs were readily cleaved by the cyclic dinucleotide PDE and oligoribonucleases (Orn). Cleavage of the fluorescent probes led to changes in fluorescence intensities. Our results suggest that these fluorescent analogs can be used to monitor the activity of cyclic dinucleotide PDEs in real time and that these probes could facilitate the identification of inhibitors of cyclic dinucleotide PDEs. Additionally these probes could be used to profile the activity of expressed PDEs and Orns.

c-di-AMP signaling regulates a myriad of physiological processes in Gram-positive bacteria and mycobacteria. c-di-AMP synthase (DAC) is essential in many human pathogens including Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae and could become an important antibacterial drug target. In our continuing efforts to identify diverse DAC inhibitors, we uncovered hydroxybenzylidene-indolinones as new DAC inhibitors. Interestingly, these compounds also possess antibacterial activities and inhibit biofilm formation. Importantly, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis could be re-sensitized to methicillin and vancomycin, respectively, by hydroxybenzylidene-indolinones.

•Quorum sensing (QS) and estradiol effect on P. aurantiaca virulence were evaluated.•Biofilm mass and DPPIV enzyme activity were used for the evaluation.•Estradiol enhanced the growth, coaggregation, and biofilm mass of P. aurantiaca.•QS Signaling disruption may, partly, inhibit virulence of P. aurantiaca.Biofilm formation and dipeptidyl peptidase IV (DPPIV) enzyme activity contribute to the virulence of oral bacteria, and these virulence factors are partly regulated by quorum sensing signaling system. We recently demonstrated that estradiol regulates growth properties and DPPIV activity of Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens. Here, we examined the DPPIV dependency of biofilm formation of Prevotella aurantiaca. Three strains (two clinical strains AHN 37505 and 37552 and the type strain CCUG 57723) were incubated in three estradiol concentrations (30, 90, and 120 nmol/L). Regulation of DPPIV activity, biofilm and fimbria formation, and coaggregation of bacterial strains were analyzed after incubation with four concentrations (10 nM, 100 nM, 1 μM, 10 μM) of dihydroxy-2,3-pentaedione (DPD), the universal precursor of autoinducer -2 (AI-2), and analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) for 24 h. Estradiol enhanced the planktonic growth, coaggregation, and biofilm formation of P. aurantiaca strains. The whole cell extract of AHN 37505 had the highest DPPIV activity, followed by CCUG 57723 and AHN 37552. Inhibition of DPPIV activity with di-isopropylfluorophosphate suppressed the effect of estradiol on biofilm formation. At 100 nM and 10 μM concentrations of DPD, butyl DPD, and isobutyl DPD, biofilm formation of P. aurantiaca was significantly inhibited. Fimbriae formation was enhanced up to concentrations of 100 nM and 1 μM followed by a significant inhibition at higher concentrations of DPD and all analogs. A slight but significant inhibitory effect of DPD and analogs on DPPIV activity was observed. Our results indicate that DPPIV plays a key role in the estradiol-regulated biofilm formation of P. aurantiaca. Quorum sensing autoinducer DPD and C1-alkyl analogs could inhibit biofilm-related virulence of P. aurantiaca.

Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.

Bacteria utilize nucleotide-based second messengers to regulate a myriad of physiological processes. Cyclic dinucleotides have emerged as central regulators of bacterial physiology, controlling processes ranging from cell wall homeostasis to virulence production, and so far over thousands of manuscripts have provided biological insights into c-di-NMP signaling. The development of small molecule inhibitors of c-di-NMP signaling has significantly lagged behind. Recent developments in assays that allow for high-throughput screening of inhibitors suggest that the time is right for a concerted effort to identify inhibitors of these fascinating second messengers. Herein, we review c-di-NMP signaling and small molecules that have been developed to inhibit cyclic dinucleotide-related enzymes.

C-di-AMP synthases are essential in several bacteria, including human pathogens; hence these enzymes are potential antibiotic targets. However, there is a dearth of small molecule inhibitors of c-di-AMP metabolism enzymes. Screening of 2000 known drugs against DisA has led to the identification of suramin, an antiparasitic drug as potent inhibitor of c-di-AMP synthase.

A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

•G-quadruplex interactive ligands easily prepared in two steps.•Alkyne-DMZ analogues inhibit c-Myc expression.•Alkyne-DMZ analogues inhibit various cancer cell lines.G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents.

Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250 000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.

C-di-AMP synthases are essential in several bacteria, including human pathogens; hence these enzymes are potential antibiotic targets. However, there is a dearth of small molecule inhibitors of c-di-AMP metabolism enzymes. Screening of 2000 known drugs against DisA has led to the identification of suramin, an antiparasitic drug as potent inhibitor of c-di-AMP synthase.

Bacteria utilize nucleotide-based second messengers to regulate a myriad of physiological processes. Cyclic dinucleotides have emerged as central regulators of bacterial physiology, controlling processes ranging from cell wall homeostasis to virulence production, and so far over thousands of manuscripts have provided biological insights into c-di-NMP signaling. The development of small molecule inhibitors of c-di-NMP signaling has significantly lagged behind. Recent developments in assays that allow for high-throughput screening of inhibitors suggest that the time is right for a concerted effort to identify inhibitors of these fascinating second messengers. Herein, we review c-di-NMP signaling and small molecules that have been developed to inhibit cyclic dinucleotide-related enzymes.