Stafiba: A STAT5-Selective Small-Molecule Inhibitor.

The transcription factors STAT5a and STAT5b are constitutively active in many human tumors. Combined inhibition of both STAT5 proteins is a valuable approach with promising applications in tumor biology. We recently reported resorcinol bisphosphate as a moderately active inhibitor of the protein-protein interaction domains, the SH2 domains, of both STAT5a and STAT5b. Here, we describe the development of resorcinol bisphosphate to Stafiba, a phosphatase-stable inhibitor of STAT5a and STAT5b with activity in the low micromolar concentration range. Our data provide insights into the structure-activity relationships of resorcinol bisphosphates and the corresponding bisphosphonates for use as inhibitors of both STAT5a and STAT5b.

Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group.

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.

Morphological Profiling Identifies the Motor Protein Eg5 as Cellular Target of Spirooxindoles.

Oxindoles and iso-oxindoles are natural product-derived scaffolds that provide inspiration for the design and synthesis of novel biologically relevant compound classes. Notably, the spirocyclic connection of oxindoles with iso-oxindoles has not been explored by nature but promises to provide structurally related compounds endowed with novel bioactivity. Therefore, methods for their efficient synthesis and the conclusive discovery of their cellular targets are highly desirable. We describe a selective RhIII -catalyzed scaffold-divergent synthesis of spirooxindole-isooxindoles and spirooxindole-oxindoles from differently protected diazooxindoles and N-pivaloyloxy aryl amides which includes a functional group-controlled Lossen rearrangement as key step. Unbiased morphological profiling of a corresponding compound collection in the Cell Painting assay efficiently identified the mitotic kinesin Eg5 as the cellular target of the spirooxindoles, defining a unique Eg5 inhibitor chemotype.

Asymmetrically Substituted m-Terphenyl Phosphates Inhibit the Transcription Factor STAT5a.

We recently presented Stafia-1 as the first chemical entity that inhibits the transcription factor STAT5a with selectivity over the highly homologous STAT5b. Stafia-1, which was identified from a series of symmetrically substituted m-terphenyl phosphates, binds to the interface between the SH2 domain and the linker domain of STAT5a. Here, we outline a synthetic strategy for the synthesis of asymmetrically substituted m-terphenyl phosphates, which can be tailored to address their asymmetric STAT5a binding site in a more specific manner. The asymmetrically substituted m-terphenyl phosphate with the highest activity against STAT5a was converted to a phosphatase-stable monofluoromethylene phosphonate. The synthetic methodology and activity analysis described here provide first insights into the structure-activity relationships of m-terphenyl phosphates for use as selective STAT5a inhibitors.

Effect of Amino Acid Substitutions on 70S Ribosomal Binding, Cellular Uptake, and Antimicrobial Activity of Oncocin Onc112.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high-priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram-negative and Gram-positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram-negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off-target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

Templated Generation of a Bcl-xL Inhibitor by Isomer-Free SPAAC Based on Azacyclonon-5-yne.

High-affinity inhibitors of large protein-protein interactions often have a high molecular weight, which compromises their cell permeability and oral bioavailability. We recently presented isomer-free, strain-promoted azide-alkyne cycloaddition (iSPAAC) as a method by which to generate large, chemically uniform bioactive molecules inside living cells from two smaller components with higher cell permeability. Here, we present the synthesis of Fmoc-protected azacyclonon-5-yne (Fmoc-ACN) as the first cyclononyne suitable for iSPAAC. ACN facilitated the structure-guided development of a single-digit micromolar triazole inhibitor of the protein-protein interaction domain of the antiapoptotic protein Bcl-xL . Inhibitor formation in aqueous buffer at 37 °C, templated by the target protein Bcl-xL , proceeded 2800 times faster than the reaction between Fmoc-ACN and benzyl azide under standard conditions in acetonitrile. Our data demonstrate the utility of cyclononynes for iSPAAC and their potential for achieving vastly accelerated templated reactions in aqueous environments.

The Selectivity of Fosfosal for STAT5b over STAT5a is Mediated by Arg566 in the Linker Domain.

Fosfosal is the O-phosphorylated derivative of salicylic acid, with documented clinical use as a prodrug for the treatment of inflammatory diseases. We recently discovered that fosfosal itself inhibits the protein-protein interaction domain, the SH2 domain, of the tumor-related transcription factor STAT5b. Here, we demonstrate that fosfosal is selective for STAT5b over its close homologue STAT5a. This selectivity is mediated by the STAT5b residue Arg566, located in the SH2 domain-adjacent linker domain. Our data provide further evidence for the role of the STAT linker domain in determining the activity of small molecules against the SH2 domain. We present a refined binding model for fosfosal and STAT5b, which can serve as the basis for the development of fosfosal-based STAT5b inhibitors.

Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments.

We present a new approach for the identification of inhibitors of phosphorylation-dependent protein-protein interaction domains, in which phenolic fragments are adapted by in silico O-phosphorylation before docking-based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product-derived phenolic fragments, which were virtually O-phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia-1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia-1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small-molecule inhibitor.

ATP Inhibits the Transcription Factor STAT5b.

Although naturally occurring low-molecular-weight compounds have many known roles within the cell, these do not usually involve the direct inhibition of protein-protein interactions. Based on the results of high-throughput screening of a library of bioactive compounds and neurotransmitters, we report here that the four nucleoside triphosphates ATP, GTP, CTP and UTP inhibit the SH2 domain of the tumor-related transcription factor STAT5b. ATP and GTP are the most active nucleoside triphosphates and show specificity for STAT5b over STAT5a, STAT3, STAT6 and the p53-binding protein HDM2. As the inhibition constant of ATP against STAT5b is significantly lower than published values for the intracellular ATP concentration, our data suggest that ATP might inhibit the protein-protein interactions of STAT5b in living cells.

Poloxin-2HT+: changing the hydrophobic tag of Poloxin-2HT increases Plk1 degradation and apoptosis induction in tumor cells.

We report the hydrophobically-tagged Plk1 PBD inhibitor Poloxin-2HT+, which selectively degrades the tumor target Plk1 and induces apoptosis in human tumor cells with higher potency than the hydrophobically-tagged inhibitor Poloxin-2HT. Our data provide further evidence that hydrophobically tagged inhibitors of protein-protein interactions can target and destroy disease-relevant proteins.

iSPAAC: Isomer-Free Generation of a Bcl-xL -Inhibitor in Living Cells.

Strain-promoted azide-alkyne cycloadditions (SPAAC) have proven extremely useful for labeling of biomolecules, but typically produce isomeric mixtures. This is not appropriate for the formation of bioactive molecules in living cells. Here, the first use of SPAAC for the isomer-free synthesis of a bioactive molecule is reported both in vitro and inside cultured cells. We developed the symmetrical cyclooctyne SYPCO and used it for the generation of a chemically uniform triazole inhibitor of protein-protein interactions mediated by Bcl-xL via isomer-free SPAAC (iSPAAC). Tumor cells treated with the reactants of the iSPAAC reaction contained higher concentrations of triazole, and displayed higher apoptosis levels, than cells treated with pre-synthesized triazole. We envision iSPAAC as a broadly applicable method for modulating intracellular targets with organic molecules with molecular weights prohibitively large for cellular uptake, via smaller and thus more cell-permeable components.

Selective Degradation of Polo-like Kinase 1 by a Hydrophobically Tagged Inhibitor of the Polo-Box Domain.

Hydrophobic tagging (HT) of bioactive compounds can induce target degradation via the proteasomal pathway. The first application of hydrophobic tagging to an existing inhibitor of protein-protein interactions is now presented. We developed Poloxin-2HT by fusing an adamantyl tag to Poloxin-2, an inhibitor of the polo-box domain of the protein kinase Plk1, which is a target for tumor therapy. Poloxin-2HT selectively reduced the protein levels of Plk1 in HeLa cells and had a significantly stronger effect on cell viability and the induction of apoptosis than the untagged PBD inhibitor Poloxin-2. The change in cellular phenotype associated with the addition of the hydrophobic tag to Poloxin-2 demonstrated that Poloxin-2HT targets Plk1 in living cells. Our data validate hydrophobic tagging of selective inhibitors of protein-protein interactions as a novel strategy to target and destroy disease-relevant proteins.

A small-molecule screen identifies the antitrypanosomal agent suramin and analogues NF023 and NF449 as inhibitors of STAT5a/b.

The transcription factor STAT5a is a potential target for tumor therapy. We present a fluorescence polarization-based, high-throughput screen of chemical libraries containing natural products and known bioactive molecules, for the identification of small-molecule inhibitors of the STAT5a SH2 domain. This screen identified suramin, a drug used to treat African trypanosomiasis, and its analogues NF023 and NF449 as inhibitors of the SH2 domains of STAT5a/b. Our data extend the known in vitro targets of suramin and analogues to include members of the STAT protein family.

Halogen-substituted catechol bisphosphates are potent and selective inhibitors of the transcription factor STAT5b.

The transcription factor STAT5b is an antitumor target. Recently, we presented the small molecules Stafib-1 and Stafib-2 as potent, selective inhibitors of the STAT5b SH2 domain. Here we report that halogen substitutions on the terminal phenyl ring of Stafib-1 and a close derivative are tolerated and specificity over the STAT5a SH2 domain is maintained, albeit with a slight reduction in activity. Our data demonstrate that the synthetic methodology used for generating Stafib-1 and Stafib-2 can be utilized to synthesize a small library of halogen-substituted derivatives, and extend the panel of catechol bisphosphate-based submicromolar and selective STAT5b inhibitors.

Inhibition of Protein-Protein Interactions: New Options for Developing Drugs against Neglected Tropical Diseases.

Wake up! Sleeping sickness and Chagas disease are neglected tropical diseases caused by trypanosome infections. Small molecules that disrupt a crucial protein-protein interaction in the parasites offer a new approach to drug development for these diseases.

Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1.

Polo-like kinase 1 (Plk1), a key player in mitosis, is overexpressed in a wide range of tumor types and has been validated as a target for tumor therapy. In addition to its N-terminal kinase domain, Plk1 harbors a C-terminal protein-protein interaction domain, referred to as the polo-box domain (PBD). Because the PBD is unique to the five-member family of polo-like kinases, and its inhibition is sufficient to inhibit the enzyme, the Plk1 PBD is an attractive target for the inhibition of Plk1 function. Although peptide-based inhibitors are invaluable tools for elucidating the nature of the binding interface, small molecules are better suited for the induction of mitotic arrest and apoptosis in tumor cells by Plk1 inhibition. This review describes the considerable progress that has been made in developing small-molecule and peptide-based inhibitors of the Plk1 PBD.

Development of Bifunctional Inhibitors of Polo-Like Kinase†1 with Low-Nanomolar Activities Against the Polo-Box Domain.

Polo-like kinase 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BI 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.

Ribosomal binding and antibacterial activity of ethylene glycol-bridged apidaecin Api137 and oncocin Onc112 conjugates.

Recent surveillance data on antimicrobial resistance predict the beginning of the post-antibiotic era with pan-resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline-rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram-negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin-type) or the assembly of the 50S subunit (apidaecin-type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112-conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)-carboxyfluorescein-labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112-conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP-susceptible and -resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin- and oncocin-type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

PYRROC: the first functionalized cycloalkyne that facilitates isomer-free generation of organic molecules by SPAAC.

We present the concept, synthesis, and kinetic characterization of PYRROC as the first functionalized cycloalkyne which cannot form isomers in the reaction with azides. In aqueous buffer, PYRROC displays unprecedented rate accelerations in SPAAC of three to four orders of magnitude, leading to rate constants exceeding 400 M(-1) s(-1).

Nanomolar inhibitors of the transcription factor STAT5b with high selectivity over STAT5a.

Src homology 2 (SH2) domains play a central role in signal transduction. Although many SH2 domains have been validated as drug targets, their structural similarity makes development of specific inhibitors difficult. The cancer-relevant transcription factors STAT5a and STAT5b are particularly challenging small-molecule targets because their SH2 domains are 93% identical on the amino acid level. Here we present the natural product-inspired development of the low-nanomolar inhibitor Stafib-1, as the first small molecule which inhibits the STAT5b SH2 domain (K(i)=44 nM) with more than 50-fold selectivity over STAT5a. The binding site of the core moiety of Stafib-1 was validated by functional analysis of point mutants. A prodrug of Stafib-1 was shown to inhibit STAT5b with high selectivity over STAT5a in tumor cells. Stafib-1 provides the first demonstration that naturally occurring SH2 domains with more than 90% sequence identity can be selectively targeted with small organic molecules.