The loading acyltransferase (AT) domains of modular polyketide synthases (PKSs) control the choice of starter units incorporated into polyketides and are therefore attractive targets for the engineering of modular PKSs. Here, we report the structural and biochemical characterizations of the loading AT from avermectin modular PKS, which accepts more than 40 carboxylic acids as alternative starter units for the biosynthesis of a series of congeners. This first structural analysis of loading ATs from modular PKSs revealed the molecular basis for the relaxed substrate specificity. Residues important for substrate binding and discrimination were predicted by modeling a substrate into the active site. A mutant with altered specificity toward a panel of synthetic substrate mimics was generated by site-directed mutagenesis of the active site residues. The hydrolysis of the N-acetylcysteamine thioesters of racemic 2-methylbutyric acid confirmed the stereospecificity of the avermectin loading AT for an S configuration at the C-2 position of the substrate. Together, these results set the stage for region-specific modification of polyketides through active site engineering of loading AT domains of modular PKSs.
3905 Poster Board III-841 Mutation of Janus Associated Kinase 2 (JAK2) at amino acid 617 and the resulting constitutively active JAK2V617F enzyme has been linked to the pathogenesis of myeloproliferative neoplasms (MPN). Fms-like Kinase 3 (FLT3) is a receptor tyrosine kinase expressed by immature hematopoietic cells. FLT3 abnormalities are present in ∼30% of acute myelogenous leukemias (AML). SB1518 is a potent ATP-competitive inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) that is selective for JAK2 inhibition compared to JAK1 and JAK3 (58 and 24 fold, respectively). It also potently inhibits FLT3 (IC50 = 22 nM) and its mutant D835Y (IC50 = 6 nM). SB1518 inhibits proliferation of human leukemia and lymphoma cell lines dependent on either JAK2 or FLT3 activation (IC50 =35-240 nM), and has antitumor activity in nude mouse models of FLT3- (MV4-11) or JAK2-dependent (BaF3-JAK2V617F) leukemia. Based on these encouraging data, SB1518 is being evaluated in subjects with acute and chronic myeloid diseases in a Phase I dose escalation study. Objectives of the trial are to determine the safety, tolerability and PK/PD profile of SB1518 when administered orally once daily continuously in 28-day cycles. Thirty-six patients have been enrolled to date and treated at 6 dose levels, from 100-600 mg daily: 31/36 myelofibrosis (MF), 5/36 AML. Median number of prior therapies was 2. At 600 mg, 3 of 6 patients experienced dose-limiting toxicity (GI symptoms requiring drug interruption). All 3 recovered and restarted SB1518 at a reduced dose. The most common related adverse events have been diarrhea 33% (4% Grade 3), nausea 13% (all Grade 1/2), and thrombocytopenia 4% (all Grade 3/4). Four deaths have occurred due to disease progression or intercurrent illness. To date, twenty-two patients remain on study and enrollment is on-going. Thirteen patients have been treated for ≥6 months and 3 ≥1 year. 21 MF patients are evaluable for response: 7/17 (41%) with palpable splenomegaly had a decrease in spleen size by physical exam of ≥ 35%, of which 4 (24%) had a decrease of ≥50%. SB1518 was rapidly absorbed with Tmax of 3-5 hours and mean elimination half-life of 2-3 days. There was no drug accumulation after repeated cycles of administration. Pharmacologically active levels were achieved at the starting dose level of 100 mg based on target efficacy biomarker assessment. Measurement of pSTAT3 and pSTAT5 in fixed whole blood and lysed peripheral blood mononuclear cells on the first day of dosing showed inhibition of pSTAT3 and pSTAT5 4-6 hours post dose at all dose levels. In conclusion, SB1518 was well tolerated at doses up to 500 mg daily in advanced patients with MF and AML, and shows promising clinical activity in MF patients with splenomegaly. Disclosures: Wood: S*BIO PTE LTD: Employment. Ethirajulu:S*BIO PTE LTD: Employment. Lowe:S*BIO PTE LTD: Consultancy. Zhu:S*BIO PTE LTD: Employment.
The allylation of aromatic and aliphatic aldehydes with allyltrichlorosilanes has been catalyzed with a new Lewis base organocatalyst, 1,1'-biscarboline N,N'-dioxide with high enantioselectivities of up to 99% for 4-methoxybenzaldehyde and 97% ee for cycloformaldehyde, respectively. In total, 13 heteroaryl and aliphatic aldehydes were tested.
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