The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia.

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC(50)=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC(50) 1 µM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

A model of activation of the protein tyrosine phosphatase SHP-2 by the human leptin receptor.

Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.

Apolipoprotein A-I binds and inhibits the human antibacterial/cytotoxic peptide LL-37.

The antibacterial and cytotoxic activity of the human cathelicidin peptide LL-37 is inhibited by plasma. Because LL-37 does not undergo rapid degradation in human plasma, we postulated that this inhibition results from binding of LL-37 to unidentified proteins. An LL-37 binding plasma protein has now been isolated by affinity chromatography. SDS-polyacrylamide gel electrophoresis of proteins that bound to an LL-37 column revealed one band with a molecular mass of about 26 kDa, and amino acid sequence analysis identified the protein as apolipoprotein A-I (apoA-I). Biomolecular interaction analysis using surface plasmon resonance showed that LL-37 and isolated apoA-I bind with an apparent Kd in the low micromolar range. 50 microM of apoA-I inhibits the antibacterial activity of 50 microM LL-37 by about 50% of the inhibition exhibited by plasma. In addition, anti-apoA-I IgG completely blocks the plasma inhibition of LL-37 antibacterial activity up to a peptide concentration of 25 microM and blocks most of the plasma inhibition at higher LL-37 concentrations. These results indicate that apoA-I is the main LL-37 binding protein in human plasma and may work as a scavenger of LL-37, thus suggesting a novel mechanism involved in the regulation of a cathelicidin peptide.

Alanine screening mutagenesis establishes tyrosine 60 of bovine insulin-like growth factor binding protein-2 as a determinant of insulin-like growth factor binding.

The determinants of insulin-like growth factor (IGF) binding to its binding proteins (IGFBPs) are poorly characterized in terms of important residues in the IGFBP molecule. We have previously used tyrosine iodination to implicate Tyr-60 in the IGF-binding site of bovine IGFBP-2 (Hobba, G. D., Forbes, B. E., Parkinson, E. J., Francis, G. L., and Wallace, J. C. (1996) J. Biol. Chem. 271, 30529-30536). In this report, we show that the mutagenic replacement of Tyr-60 with either Ala or Phe reduced the affinity of bIGFBP-2 for IGF-I (4.0- and 8.4-fold, respectively) and for IGF-II (3.5- and 4.0-fold, respectively). Although adjacent residues Val-59, Thr-61, Pro-62, and Arg-63 are well conserved in IGFBP family members, Ala substitution for these residues did not reduce the IGF affinity of bIGFBP-2. Kinetic analysis of the bIGFBP-2 mutants on IGF biosensor chips in the BIAcore instrument revealed that Tyr-60 --> Phe bIGFBP-2 bound to the IGF-I surface 3.0-fold more slowly than bIGFBP-2 and was released 2.6-fold more rapidly than bIGFBP-2. We therefore propose that the hydroxyl group of Tyr-60 participates in a hydrogen bond that is important for the initial complex formation with IGF-I and the stabilization of this complex. In contrast, Tyr-60 --> Ala bIGFBP-2 associated with the IGF-I surface 5.0-fold more rapidly than bIGFBP-2 but exhibited an 18.4-fold more rapid release from this surface compared with bIGFBP-2. Thus both the aromatic nature and the hydrogen bonding potential of the tyrosyl side chain of Tyr-60 are important structural determinants of the IGF-binding site of bIGFBP-2.