Yaguo Zheng

Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide].

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton′s tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 × 8 × 9 × 17 Å, and the thickness of the pocket is approximately 7 Å. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a K i value of 1.4 μm, inhibited human BTK in vitro with an IC50 value of 17.2 ± 0.8 μm. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 μm. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 μg/ml (∼278 μm), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.

In vivo antioxidant activity of genistein in a murine model of singlet oxygen-induced cerebral stroke

We evaluated the in vivo antioxidant activity of genistein in a mouse model of singlet oxygen-induced cerebral stroke. Cerebral stroke was induced in male BALB/c mice through extensive microvessel damage caused by photoactivated rose bengal dye. The photoactivation of the intravenously administered rose bengal was achieved by transcranial illumination with green light. Genistein was more active than its analogs and other antioxidants that were used as control agents. At a dose of 16 mg/kg genistein administered every 6 h from 24 h prior to irradiation until 24 h after irradiation, the average size of the cerebral lesion of genistein-treated mice was significantly smaller than that in control mice treated with the carrier DMSO (8.1 +/- 1.0 mm(2) compared to 14.6 +/- 0.7 mm(2), P < 0.001). Our findings provide experimental evidence that genistein could be useful for the prevention of cerebral stroke and other pathological conditions caused by reactive oxygen species.

The Anti-leukemic Bruton's Tyrosine Kinase Inhibitor α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl) Propenamide (LFM-A13) Prevents Fatal Thromboembolism

Abstract The leflunomide metabolite analog α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruto ns tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRγ-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 2.8 μM. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100mg/kg. At nontoxic dose levels, LFM-A13 prolonged the tail bleeding times of mice and improved event-free survival in two mouse models of agonist-induced invariably fatal pulmonary thromboembolism. To our knowledge, LFM-A13 is the first anti-thrombotic agent which prevents platelet aggregation by inhibiting BTK.

Rational design of potent and selective EGFR tyrosine kinase inhibitors as anticancer agents.

Increasing knowledge of the structure and function of the Epidermal Growth Factor Receptor (EGFR) subfamily of tyrosine kinases, and of their role in the initiation and progression of various cancers has led to the search for inhibitors of signaling molecules that may prove to be important in cancer therapy. The complex nature of EGFR biology allows for potential opportunities for EGFR inhibitors in a number of areas of cancer therapy, including proliferative, angiogenic, invasive, and metastatic aspects. Different approaches have been used to target either the extracellular ligand-binding domain of the EGFR or the intracellular tyrosine kinase region that results in interference with its signaling pathways that modulate cancer-promoting responses. Examples of these include a number of monoclonal antibodies, immunotoxins and ligand-binding cytotoxic agents that target the extracellular ligand binding region of EGFR, and small molecule inhibitors that target the intracellular kinase domain and act by interfering with ATP binding to the receptor. During the past 3 years, significant progress has been made towards the identification of new structural classes of small molecule inhibitors that show high potency and specificity towards EGFR. The search for new small molecules that inhibit kinases has included traditional approaches like the testing of natural products, random screening of chemical libraries, the use of classical structure-activity-relationship studies, and the incorporation of structure-based drug design and combinatorial chemistry techniques. There has been a significant improvement in the development of selective EGFR inhibitors with the use of a structure-based design approach employing a homology model of the EGFR kinase domain. Molecular modeling procedures have been used to generate novel molecules that are complementary in shape and electrostatics to the EGFR kinase domain topography. This review focuses on some examples of the successful use of this method.

Three leflunomide metabolite analogs

The title compounds, 1-cyano-2-hydroxy-N-[4-(methylsulfonyl)phenyl]but-2-enamide, C(12)H(12)N(2)O(4)S, PHI492, 1-cyano-2-hydroxy-N-[3-(methylsulfonyl)phenyl]but-2-enamide, C(12)H(12)N(2)O(4)S, PHI493, and N-[3-bromo-4-(trifluoromethoxy)phenyl]-1-cyano-2-hydroxybut-2-e namide , C(12)H(8)BrF(3)N(2)O(3), PHI495, are potent inhibitors of Brutons tyrosine kinase (BTK). The molecular structures of these compounds are similar and they display similar hydrogen-bonding networks and crystal packing. Examination of the crystal-packing interaction in the three compounds reveals an alternating direction of adjacent molecules in the crystalline lattice due to intermolecular cyano-amide hydrogen bonding. PHI492, a positional isomer of PHI493, does not form intermolecular O-H.O hydrogen bonds between molecules and crystallizes in a space group different from that of PHI493 and PHI495. The aromatic ring and the amide group of each molecule form a conjugated pi-system which ensures planarity, with further stabilization gained from intramolecular O-H.O hydrogen bonds.

Five analogs of the active metabolite of leflunomide

The title compounds, 2-cyano-3-hydroxy-N-(4-bromo-phenyl)but-2-enamide, C 11 H 9 BrN 2 O 2 (LFM-A1), 2-cyano-3-hydroxy-N-(2-fluorophenyl)but-2-enamide, C 11 H 9 FN 2 O 2 (LFM-A7), 2-cyano-3-hydroxy-N-(3-bromophenyl)but-2-enamide, C 11 H 9 BrN 2 O 2 (LFM-A9), 2-cyano-3 -hydroxy-N-(3 -chlorophenyl)but-2-enamide, C 11 H 9 ClN 2 O 2 (LFM-A10), and 2-cyano-3-hydroxy-N-(3-fluorophenyl)but-2-enamide, C 11 H 9 FN 2 O 2 (LFM-A11), are analogs of A77 1726, the active metabolite of the immunosupressive drug leflunomide, which is known to act in part by inhibiting the tyrosine kinase epidermal growth factor receptor (EGFR) [Mattar, Kochhar, Bartlett, Bremer & Finnegan (1993). FEBS Lett. 334, 161-164]. The molecular structures of the title compounds are very similar and they display similar crystal packing and hydrogen-bonding networks. All five molecules are approximately planar; the dihedral angles between the phenyl ring and the plane defined by the N-C-C=C-CH 3 group are 4.8(8)° for LFM-A1, 12.5 (2)° for LFM-A7, 6.2(6)° for LFM-A9, 5.5 (3)° for LFM-A10 and 4.4(3)° for LFM-A11. The intramolecular hydrogen bond between the O atoms observed in all the compounds locks them into a planar conformation and may contribute to a conformation which is favorable for binding the shallow ATP-binding pocket of EGFR.