Lisa Tuel-Ahlgren

Rational design and synthesis of phenethyl-5-bromopyridyl thiourea derivatives as potent non-nucleoside inhibitors of HIV reverse transcriptase

Abstract A series of novel phenethylthiazolylthiourea (PETT) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of HIV reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket. The structure-based design and synthesis of these new PETT derivatives were complemented by biological assays of their anti-HIV activity. Modeling studies for rational drug design included the construction of a composite NNI binding pocket from nine RT-NNI crystal structures, the analyses of surface complementarity between NNI and RT, and application of K i calculations combined with a docking procedure involving the novel PETT derivatives. The use of the composite NNI binding pocket allowed the identification and structure-based design of three promising PETT derivatives with ortho -F ( 2 ), ortho -Cl ( 3 ), and meta -F ( 5 ) substituents on the phenyl ring. These novel PETT derivatives were more active than AZT or trovirdine and showed potent anti-HIV activity with IC 50 [p24] values of 100,000.

STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress

We provide unprecedented genetic and biochemical evidence that the antiapoptotic transcription factor STAT3 serves as a substrate for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK in an ecdysone-inducible mammalian expression system results in STAT3 activation, as documented by tyrosine phosphorylation and nuclear translocation of STAT3, as well as amplified expression of several STAT3 target genes. STAT3 activation after oxidative stress (OS) is strongly diminished in DT40 chicken B-lineage lymphoma cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 domain-mutated, but not a kinase domain-mutated, syk gene into SYK-deficient DT40 cells restores OS-induced enhancement of STAT-3 activity. Thus, SYK plays an important and indispensable role in OS-induced STAT3 activation and its catalytic SH1 domain is critical for this previously unknown regulatory function. These results provide evidence for the existence of a novel mode of cytokine-independent cross-talk that operates between SYK and STAT3 pathways and regulates apoptosis during OS. We further provide experimental evidence that SYK is capable of associating with and phosphorylating STAT3 in human B-lineage leukemia/lymphoma cells challenged with OS. In agreement with a prerequisite role of SYK in OS-induced STAT3 activation, OS does not induce tyrosine phosphorylation of STAT3 in SYK-deficient human proB leukemia cells. Notably, inhibition of SYK with a small molecule drug candidate prevents OS-induced activation of STAT3 and overcomes the resistance of human B-lineage leukemia/lymphoma cells to OS-induced apoptosis.

5-Alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-ones as potent non-nucleoside reverse transcriptase inhibitors of S-DABO series

Novel dihydroalkoxybenzyloxopyrimidine (S-DABO) derivatives targeting the non-nucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been synthesized using a novel computer model for the NNI binding pocket and tested for their RT inhibitory activity in cell-free assays using purified recombinant HIV RT as well as for their anti-HIV activity in HTL VIIIB-infected peripheral blood mononuclear cells. Our computational approach allowed the identification of several ligand derivatization sites for the generation of more potent S-DABO derivatives. Our lead S-DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (compound 3), elicited potent anti-HIV activity with an IC50 value of less than 1nM for inhibition of HIV replication without any evidence of cytotoxicity and an unprecedented selectivity index of > 100,000.

Engagement of the CD19 receptor on human B-lineage leukemia cells activates LCK tyrosine kinase and facilitates radiation-induced apoptosis.

As presently reported, both ionizing radiation and engagement of the CD19 receptor are capable of inducing apoptosis in B-lineage acute lymphoblastic leukemia (ALL) cells. In both instances, activation of tyrosine kinases appears to be a proximal and mandatory step, since it can be prevented by the tyrosine kinase inhibitor genistein. This common biochemical signaling pathway involves the rapid activation of the Src family tyrosine kinase LCK (p56lck), which is physically associated with the CD19 receptor, and enhanced tyrosine phosphorylation of multiple substrates leading to stimulation of phosphoinositide turnover, and activation of protein kinase C. Importantly, engagement of the CD19 receptor promoted radiation-induced apoptosis in radiation-resistant B-lineage ALL cells in a cell type-specific fashion. Our results prompt the hypothesis that clonogenic B-lineage ALL blasts with an inherent or acquired resistance to radiation could be radiosensitized in clinical settings using anti-CD19 MoAb B43 or its homoconjugate as adjuncts.

ELECTROMAGNETIC FIELD-INDUCED STIMULATION OF BRUTON'S TYROSINE KINASE

Here we present evidence that exposure of DT40 lymphoma B-cells to low energy electromagnetic fields (EMF) results in activation of phospholipase C-γ 2 (PLC-γ2), leading to increased inositol phospholipid turnover. PLC-γ2 activation in EMF-stimulated cells is mediated by stimulation of the Bruton’s tyrosine kinase (BTK), a member of the Src-related TEC family of protein tyrosine kinases, which acts downstream of LYN kinase and upstream of PLC-γ2. B-cells rendered BTK-deficient by targeted disruption of thebtk gene did not show enhanced PLC-γ2 activation in response to EMF exposure. Introduction of the wild-type (but not a kinase domain mutant) human btk gene into BTK-deficient B-cells restored their EMF responsiveness. Thus, BTK exerts a pivotal and mandatory function in initiation of EMF-induced signaling cascades in B-cells.

Polo‐like‐kinase 1 (PLK1) as a molecular target to overcome SYK‐mediated resistance of B‐lineage acute lymphoblastic leukaemia cells to oxidative stress

SYK tyrosine kinase has emerged as a master regulator of cellular resistance to oxidative stress (OS) by mediating the activation of the anti‐apoptotic nuclear factor κB and phosphatidylinositol‐3 kinase/AKT pathways after OS exposure. Here, we present unprecedented experimental evidence that polo‐like kinase 1 (PLK1) is the upstream regulator of SYK in B‐lineage acute lymphoblastic leukaemia (ALL) cells. Selective inhibition of PLK‐1 with the leflunomide metabolite analogue α‐cyano‐β‐hydroxy‐β‐methyl‐N‐[4‐(trifluoromethoxy) phenyl]‐propenamide/LFM‐A12 abolished the resistance of B‐lineage ALL cells to OS by preventing the activation of the anti‐apoptotic SYK signal transduction pathway. Notably, LFM‐A12 treatments at non‐cytotoxic concentrations resulted in marked augmentation of clonogenic death in resistant human B‐lineage ALL cell lines challenged with OS. Further, LFM‐A12 augmented OS‐induced apoptosis of chemotherapy‐resistant primary leukaemic cells from relapsed B‐lineage ALL patients in vitro and markedly potentiated the in vivo anti‐leukaemic activity of total body irradiation (TBI) against leukaemia‐initiating cells in severe combined immunodeficient mouse xenograft models of B‐lineage ALL. This study is the first to identify PLK1 as a regulator of SYK tyrosine kinase and a molecular target to overcome SYK‐mediated resistance of B‐lineage ALL cells to OS.

Expression of BCR-ABL, E2A-PBX1, and MLL-AF4 fusion transcripts in newly diagnosed children with acute lymphoblastic leukemia: a Children's Cancer Group initiative.

We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2A-PBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Childrens Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.

N-[2-(4-methylphenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea as a potent inhibitor of NNRTI-resistant and multidrug-resistant human immunodeficiency virus type 1.

The composite non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket model was used to study a number of thiourea analogues with different substitutions at the 4-phenyl position including N-[2-(4-methylphenyl)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea (compound HI-244), which inhibited recombinant RT better than trovirdine or compound HI-275 with an unsubstituted phenyl ring. HI-244 effectively inhibited the replication of HIV-1 strain HTLVIIIB in human peripheral blood mononuclear cells with an IC50 value of 0.007 μM, which is equal to the IC50 value of trovirdine. Notably, HI-244 was 20 times more effective than trovirdine against the multidrug-resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74 V, 41L and 215Y) and seven times more potent than trovirdine against the NNRTI- resistant HIV-1 strain A17 with a Y181C mutation.

Role of Hydroxyl Radicals in Radiation-Induced Activation of Lyn Tyrosine Kinase in Human B-Cell Precursors

Here we show that exposure of human B-cell precursors to gamma-rays stimulates the enzymatic activity of the Src protooncogene family protein tyrosine kinase LYN. LYN activation in irradiated cells is not triggered by DNA damage or a nuclear signal since gamma-rays effectively stimulated LYN kinase in enucleated B-cell precursors as well. LYN activation in irradiated cells was abrogated by presence of the OH* radical scavenger dimethylsulfoxide and exposure of intact or enucleated B-cell precursors to chemically generated OH* radicals instead of gamma-rays also triggered LYN kinase activation and enhanced tyrosine phosphorylation of multiple electrophoretically distinct protein substrates. Thus, OH* radicals appear to be both mandatory and sufficient for radiation-induced LYN kinase activation in irradiated B-cell precursors. We further present evidence which indicates that OH* radicals activate LYN by a novel mechanism which involves disruption of inactive LYN-LYN homodimers and monomerization of the LYN kinase after proteolytic degradation of a putative LYN-associated adapter protein through a cytoplasmic TPCK-sensitive chymotrypsin-like protease following its oxidation. LYN kinase plays a pivotal role in initiation of signal cascades that affect the proliferation, differentiation, and survival of B-cell precursors. Our results prompt the hypothesis that a growth regulatory balance might be altered in human B-cell precursors by radiation-induced stimulation of LYN kinase.

Role of Tyrosine Phosphorylation in Radiation-Induced Cell Cycle-Arrest of Leukemic B-Cell Precursors at the G2-M Transition Checkpoint

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.