Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

Photo-crosslinking of clinically relevant kinases using H89-derived photo-affinity probes.

The profiling of kinases using established proteomics techniques is hampered by their non-covalent mode-of-action. One way to overcome this caveat is the use of probes featuring photo-labelling groups that can be activated by UV irradiation to generate a reactive species that will establish a covalent bond to the enzyme. In this study we have used the well-known kinase inhibitor H89 as a lead for the development of probes for the affinity-based profiling of clinically relevant kinases. A labelling protocol was established for recombinant kinases and more complex protein mixtures using gel-based techniques. We also show that the probes act in a competitive manner with other kinase inhibitors.

A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [¹8F]FB-ML5, as a molecular probe for non-invasive MMP/ADAM-targeted imaging.

BACKGROUND: Numerous clinical studies have shown a correlation between increased matrix metalloproteinase (MMP)/a disintegrin and metalloproteinase (ADAM) activity and poor outcome of cancer. Various MMP inhibitors (MMPIs) have been developed for therapeutic purposes in oncology. In addition, molecular imaging of MMP/ADAM levels in vivo would allow the diagnosis of tumors. We selected the dual inhibitor of MMPs and ADAMs, ML5, which is a hydroxamate-based inhibitor with affinities for many MMPs and ADAMs. ML5 was radiolabelled with (18)F and the newly obtained radiolabelled inhibitor was evaluated in vitro and in vivo. MATERIALS AND METHODS: ML5 was radiolabelled by direct acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) for PET (positron emission tomography). The resulting radiotracer [(18)F]FB-ML5 was evaluated in vitro in human bronchial epithelium 16HBE cells and breast cancer MCF-7 cells. The non-radioactive probe FB-ML5 and native ML5 were tested in a fluorogenic inhibition assay against MMP-2, -9, -12 and ADAM-17. The in vivo kinetics of [(18)F]FB-ML5 were examined in a HT1080 tumor-bearing mouse model. Specificity of probe binding was examined by co-injection of 0 or 2.5mg/kg ML5. RESULTS: ML5 and FB-ML5 showed high affinity for MMP-2, -9, -12 and ADAM-17; indeed IC50 values were respectively 7.4 ± 2.0, 19.5 ± 2.8, 2.0 ± 0.2 and 5.7 ± 2.2 nM and 12.5 ± 3.1, 31.5 ± 13.7, 138.0 ± 10.9 and 24.7 ± 2.8 nM. Radiochemical yield of HPLC-purified [(18)F]FB-ML5 was 13-16% (corrected for decay). Cellular binding of [(18)F]FB-ML5 was reduced by 36.6% and 27.5% in MCF-7 and 16 HBE cells, respectively, after co-incubation with 10 µM of ML5. In microPET scans, HT1080 tumors exhibited a low and homogeneous uptake of the tracer. Tumors of mice injected with [(18)F]FB-ML5 showed a SUVmean of 0.145 ± 0.064 (n=6) which decreased to 0.041 ± 0.027 (n=6) after target blocking (p<0.05). Ex vivo biodistribution showed a rapid excretion through the kidneys and the liver. Metabolite assays indicated that the parent tracer represented 23.2 ± 7.3% (n=2) of total radioactivity in plasma, at 90 min post injection (p.i.). CONCLUSION: The nanomolar affinity MMP/ADAM inhibitor ML5 was successfully labelled with (18)F. [(18)F]FB-ML5 demonstrated rather low binding in ADAM-17 overexpressing cell lines. [(18)F]FB-ML5 uptake showed significant reduction in the HT1080 tumor in vivo after co-injection of ML5. [(18)F]FB-ML5 may be suitable for the visualization/quantification of diseases overexpressing simultaneously MMPs and ADAMs.

Exploring the conformational and biological versatility of ß-turn-modified gramicidin S by using sugar amino acid homologues that vary in ring size.

Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the ß-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.

Structural and biological evaluation of some loloatin C analogues.

Loloatin C is a cyclic cationic antimicrobial peptide which is active against gram positive as well as certain gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.

Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

Reciprocal chemical genetics for swift lead and target identification.

Reciprocal chemical genetics: correlation between chemical profile of in vivo phenotypes and in vitro data of potential target genes leads to swift target identification.

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

Facile synthesis and application of uniformly 13C, 15N-labeled phosphotyrosine for ligand binding studies.

The first synthesis of [U-13C, 15N] labeled phosphotyrosine is described. Preliminary studies toward the binding of phosphotyrosine to an SH2 domain have been performed by means of heteronuclear NMR.