Tyrosine kinases as new molecular targets in treatment of inflammatory disorders and leukemia.

Many protein tyrosine kinases (PTK), including Janus kinase 3 (JAK3) and Bruton's tyrosine kinase (BTK), have been recently identified as potential drug targets to treat diverse diseases including inflammation and cancer. The wealth of structural information currently available for protein kinase-inhibitor complexes facilitates the structure-based design of novel kinase inhibitors. In this report, we discuss the structural basis of protein kinase inhibitor design and the common binding features of small molecule kinase inhibitors including pyridinyl imidazoles, purines, oxindoles, anilinoquinazolines and isoquinalines.

Phenyl phosphoramidate derivatives of stavudine as anti-HIV agents with potent and selective in-vitro antiviral activity against adenovirus.

Adenoviruses are responsible for a broad range of clinical diseases that may be associated with high mortality, including pneumonia, hepatitis, encephalitis, hemorrhagic cystitis, nephritis, and gastroenteritis in immunocompromised patients, including HIV-infected individuals. Here we report the identification of halo-substituted stavudine phenyl phosphoramidate derivatives as a new class of dual-function anti-HIV agents with potent and selective anti-adenovirus (ADV) activity. We examined the investigational stavudine phenyl phosphoramidate derivative stampidine and 12 structurally similar stavudine derivatives for anti-ADV activity.

Antiretroviral spermicide WHI-07 prevents vaginal and rectal transmission of feline immunodeficiency virus in domestic cats.

WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model.

Stampidine is a potential nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide.

Objective: Stampidine (2,'3'-didehydro-3'-deoxythymidine-5'-(p-bromophenyl methoxy alaninyl phosphate) is a novel aryl phosphate derivative of stavudine/d4T with broad-spectrum anti-HIV activity in vitro and in vivo. This study investigated the potential utility of stampidine as a nonspermicidal microbicide.

Design: Prospective, controlled study.

Clinical development of microbicides for the prevention of HIV infection.

The HIV/AIDS pandemic continues its spread at a rate of over 15,000 new infections every day. Sexual transmission of HIV-1 is the dominant mode of this pandemic spread. For the first time since the disease emerged in the early 1980s, about half the 42 million people now living with HIV/AIDS worldwide are women.

GM-144, a novel lipophilic vaginal contraceptive gel-microemulsion.

In a systematic effort to develop a dual-function intravaginal spermicide as well as a drug delivery vehicle against sexually transmitted pathogens, a submicron particle size (30-80 nm), lipophilic and spermicidal gel-microemulsion (viz GM-144) containing the pharmaceutical excipients propylene glycol, Captex 300, Cremophor EL, Phospholipon 90G, Rhodigel, Pluronic F-68, and sodium benzoate was formulated. GM-144 completely immobilized sperm in human or rabbit semen in less than 30 seconds. Therefore, the in vivo contraceptive potency of intravaginally applied GM-144 was compared in the standard rabbit model to those of the detergent spermicide, nonoxynol-9 (N-9)-containing formulation.

Developmental toxicology studies of WHI-07, a novel nucleoside analogue-based dual-function microbicide, administered intravaginally to rabbits.

The zidovudine derivative, 5-bromo-6-methoxy-5,6-dihydro-3-azidothymidine-5'-(p-bromophenyl) methoxy alaninyl phosphate (WHI-07), is a dual-function spermicidal and anti-HIV agent with contraceptive and microbicidal activity. In previous subchronic and reproductive toxicity studies and a two-year carcinogenicity study, daily intravaginal application of 0.5 to 2.

The anti-leukemic Bruton's tyrosine kinase inhibitor alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl) propenamide (LFM-A13) prevents fatal thromboembolism.

The leflunomide metabolite analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRgamma-coupled collagen receptor signaling pathway. At low micromolar concentrations, LFM-A13 inhibited collagen-induced ultrastructural changes indicative of activation. LFM-A13 inhibited collagen (but not thrombin, TRAP-6, or ADP)-induced platelet aggregation in a concentration-dependent fashion with an IC50 value of 2.

Prevention of islet allograft rejection in diabetic mice by targeting Janus Kinase 3 with 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (JANEX-1).

Janus kinase (JAK) 3-deficient mice were not able to reject allogeneic islet allografts. The JAK3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (CAS 202475-60-3, JANEX-1, WHI-P131) prevented the rejection of islet allografts in mice with a normal JAK3 expression status. The combination of JANEX-1 and cyclosporin A (CAS 59865-13-3) was more effective than either agent alone.

Contraceptive activity of a spermicidal aryl phosphate derivative of bromo-methoxy zidovudine (compound WHI-05) in rabbits.

WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl) methoxyalaninyl phosphate, CAS 213982-93-5) is a highly promising anti-viral and spermicidal agent with clinical potential as a vaginal contraceptive which can prevent the sexual transmission of AIDS. In this study, the contraceptive activity of WHI-05 in rabbits was evaluated. Exposure of semen to WHI-05 at the time of artificial insemination inhibited pregnancy and embryo implantation by 90%.

Development and evaluation of a thermoreversible ovule formulation of stampidine, a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide.

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions.

Phenylarsonic acid compounds with broad-spectrum and potent cytotoxic activity against human cancer cells.

The in vitro cytotoxic activity profile of nine novel phenylarsonic acid (CAS 98-05-5, PAA) compounds against 17 human cancer cell lines including (a) ovarian cancer cell lines ES-2, PA-1, CAOV-3, OVCAR-3, (b) testicular cancer cell lines Ntera-2, Tera-2, N2NICP, 833K, and 64CP, (c) multiple myeloma cell lines ARH77, HS-Sultan, RPMI-8226, and U266, and (d) acute lymphoblastic leukemia (ALL) cell lines NALM-6, MOLT-3, ALL-1, and RS4; 11, was determined by the MTT assay. The lead compounds, 2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine (PHI-370) and 2-methylthio-4-(4'-phenylarsonic acid)-aminopyrimidine (PHI-380) caused apoptotic death in all 17 cancer cell lines at low micromolar concentrations, as documented by TUNEL assays and confocal laser scanning microscopy. PHI-380 was also tested and found to be very active against primary tumor cells isolated from surgical biopsy specimens of 14 patients with therapy-refractory non-small cell lung cancer, breast cancer, colon cancer, lymphoma, hepatoblastoma, or Wilm's tumor as well.

Defective expression of Bruton's tyrosine kinase in acute lymphoblastic leukemia.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that serves an essential role in B cell signaling and development. We examined the BTK expression profile of primary leukemic cells from infants with newly diagnosed acute lymphoblastic leukemia (ALL) (N = 14) and from pediatric patients with newly diagnosed (N = 10) or relapsed (N = 5) B-lineage ALL. Analysis of BTK protein and mRNA expression in the infant patient cells (N = 14) showed variable levels of BTK expression with the majority of samples having reduced to absent BTK expression.

Toxicity and pharmacokinetics of stampidine in mice and rats.

The in vivo toxicity and pharmacokinetics of stampidine (CAS 217178-62-6), an aryl phosphate derivative of stavudine (CAS 3056-17-5) under development as a new anti-human immunodeficiency virus (anti-HIV) agent, were studied in mice and rats. Stampide was very well tolerated by both mice and rats without any toxicity at cumulative dose levels > 1 g/kg. Therapeutic micromolar plasma concentrations of stampidine and its active metabolites ala-STV-MP (CAS 180076-92-0) and STV were rapidly achieved and maintained several hours after i.

Contraceptive activity of a spermicidal aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07) in rabbits.

Objective: To determine the vaginal contraceptive activity of WHI-07 in the rabbit model.

Design: Prospective, controlled study.

Setting: Center for advanced preclinical sciences.

Hypermethylation of the spleen tyrosine kinase promoter in T-lineage acute lymphoblastic leukemia.

Sequence analysis of the noncoding first exon (exon 1) of the Syk gene demonstrated the presence of a previously cloned CpG island (GenBank #Z 65706). Transient transfection analysis in Daudi cells demonstrated promoter activity (18-fold increase over parental luciferase plasmid) for a 348 bp BstXI-BsrBI fragment containing this island. This region exhibits a high GC content (approximately 75%), contains several SP1 binding sites and a potential initiator sequence, but lacks a strong TATA consensus.

Targeting JAK3 with JANEX-1 for prevention of autoimmune type 1 diabetes in NOD mice.

Here we show that Janus kinase (JAK) 3 is an important molecular target for treatment of autoimmune insulin-dependent (type 1) diabetes mellitus. The rationally designed JAK3 inhibitor JANEX-1 exhibited potent immunomodulatory activity and delayed the onset of diabetes in the NOD mouse model of autoimmune type 1 diabetes. Whereas 60% of vehicle-treated control NOD mice became diabetic by 25 weeks, the incidence of diabetes at 25 weeks was only 9% for NOD females treated with daily injections of JANEX-1 (100 mg/kg/day) from Week 10 through Week 25 (P = 0.

Synthesis and anti-HIV activity of carbamates of antiviral agent stavudine.

An efficient synthesis of carbamate analogues of the NRTI compound stavudine, has been achieved in five steps starting from commercially available thymidine. The synthesis involves conversion of thymidine into stavudine followed by condensation with carbaimidazole derivative obtained from various aromatic and heterocyclic amines in dimethylformamide solvent. The analogues thus obtained were further purified by crystallization to furnish analytically pure products.

Substituted heterocyclic thiourea compounds as a new class of anti-allergic agents inhibiting IgE/Fc epsilon RI receptor mediated mast cell leukotriene release.

Mast cell derived leukotrienes (LT's) play a vital role in pathophysiology of allergy and asthma. We synthesized various analogues of indolyl, naphthyl and phenylethyl substituted halopyridyl, thiazolyl and benzothiazolyl thioureas and examined their in vitro effects on the high affinity IgE receptor/Fc epsilon RI-mediated mast cell leukotriene release. Of the 22 naphthylethyl thiourea compounds tested, there were 7 active compounds and N-[1-(1-naphthyl)ethyl]-N'-[2-(ethyl-4-acetylthiazolyl)]thiourea (17 and 16) (IC(50)=0.

Jak3 expression and genomic sequence in pediatric acute lymphoblastic leukemia.

Janus tyrosine kinase 3 (JAK3) is one of several key regulatory enzymes in B-cell precursors which is highly conserved between multiple species. The gene for Jak3 has been mapped to human chromosome 19p12-13.1 and encompasses 23 exons.

CD95 (APO-1/FAS) deficiency in infant acute lymphoblastic leukemia: detection of novel soluble Fas splice variants.

Fas (APO-1/CD95) is a 45-kDa membrane protein which regulates apoptosis in many lymphoid cell types. In the present study, FAS expression was examined in primary leukemic cells from infants with acute lymphoblastic leukemia (ALL). The cells were resistant to apoptosis induction by an anti-FAS antibody and expressed nearly undetectable amounts of FAS protein.

Potent dual anti-HIV and spermicidal activities of novel oxovanadium(V) complexes with thiourea non-nucleoside inhibitors of HIV-1 reverse transcriptase.

We have previously demonstrated that tetrahedral bis(cyclopentadienyl)vanadium(IV) complexes and square pyramidal oxovanadium(IV) complexes of vanadium are rapid and selective spermicidal agents at low micromolar concentrations. This study investigated the potential utility of oxovanadium in combination with thiourea non-nucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) for the development of an effective dual-function anti-HIV spermicide. Two rationally designed substituted phenyl-ring containing pyridyl thiourea NNIs, N-[2-(2-chlorophenethyl)]-N(')-[2-(5-bromopyridyl)-thiourea) [1] and N-[2-(2-methoxyphenethyl)]-N(')-[2-(pyridyl)-thiourea [2] that exhibited subnanomolar IC(50) values against the drug-sensitive, drug-resistant, and multidrug-resistant strains of HIV-1, were complexed with oxovanadium.

Organic phenyl arsonic acid compounds with potent antileukemic activity.

A series of 12 organic arsonic acid compounds has been synthesized and evaluated against human B-lineage (NALM-6) and T-lineage (MOLT-3) acute lymphoblastic leukemia (ALL) cell lines. The lead compounds 2-trichloromethyl-4-[4'-(4"-phenylazo)phenylarsonic acid]aminoquinazoline (compound 19, PHI-P518; IC(50)=1.1+/-0.

Inhibition of mast cell leukotriene release by thiourea derivatives.

Mast cell derived leukotrienes (LT's) play a vital role in pathophysiology of allergy and asthma. We synthesized various analogues of indolyl, naphthyl and phenylethyl substituted halopyridyl, thiazolyl and benzothiazolyl thioureas and examined their in vitro effects on the high affinity IgE receptor/FcERI-mediated mast cell leukotriene release. Of the 22 naphthylethyl thiourea compounds tested, there were seven active compounds and N-[1-(1-naphthyl)ethyl]-N'-[2-(ethyl-4-acetylthiazolyl)]thiourea (17 and 16) (IC(50)=0.

SYK and LYN mediate B-cell receptor-independent calcium-induced apoptosis in DT-40 lymphoma B-cells.

Here, we report that the calcium ionophore ionomycin induces a massive Ca2+-dependent apoptosis in wildtype DT-40 chicken B lymphoma cells, as well as in BTK-deficient, PLCgamma2-deficient and IP3 receptor-deficient DT-40 cells, but not in LYN- or SYK-deficient DT-40 cells. Notably, the deficiency of CSK, a negative regulator of Src-family PTK, promoted ionomycin-induced apoptosis of DT-40 cells. Reconstitution of SYK-deficient cells with wild-type SYK restored the apoptotic response of the cells to ionomycin, but the expression of FYN or LCK in LYN-deficient cells did not restore the apoptotic response of LYN-deficient cells.