<div>Abstract<p>Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC<sub>50</sub> 6–35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC<sub>50</sub> 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC<sub>50</sub> 50–150 nmol/L). <i>In vivo</i>, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.</p></div>
The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of β-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of β-D-glucose, to be absolutely essential for the efficient binding of β-D-glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of β-D-glucose, exhibits an 80-fold higher apparent Km (513 mM) but a Vmax only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of β-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent Km more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of β-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation.
Abstract Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.
Munc18b is a mammalian Sec1-related protein that is abundant in epithelial cells and regulates vesicle transport to the apical plasma membrane. We constructed a homology model of Munc18b in complex with syntaxin 3 based on the crystal structure of the neuronal Sec1·syntaxin 1A complex. In this model we identified all residues in the interface between the two proteins that contribute directly to the interaction and mutagenized residues in Munc18b to alter its binding to syntaxins 1A, 2, and 3. The syntaxin-binding properties of the mutants were tested using an in vitroassay and by a co-immunoprecipitation approach employing Munc18b expressed in CHO-K1 cells. Three Munc18b variants, W28S, S42K, and E59K, were generated that are defective in binding to all three syntaxins. A fourth mutant protein, S48D, shows abolishment of syntaxin 3 interaction but binds syntaxin 2 at normal and syntaxin 1A at mildly reduced efficiency. Over-expression of Munc18b S48D inhibited transport of influenza hemagglutinin to the apical surface of Madin-Darby canine kidney II cells, which express syntaxin 2 abundantly, but not of Caco-2 cells, in which syntaxin 3 is the major apical target SNARE (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors). This suggests that, although syntaxin 3 is the main target SNARE operating in exocytic transport to the apical plasma membrane in certain epithelial cell types, syntaxin 2 may play an important role in this trafficking route in others.
The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala224) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Å (= 0.1nm) closer to the acid/base Glu217 residue, with a 3.1 Å contact between N∊2 and O∊1. The pH variation of kcat/Km for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK1 shifting from 2.22±0.03 in the wild-type to 3.19±0.03 in the mutant, and pK2 shifting from 5.99±0.02 to 6.78±0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative kcat for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower kcat/Km values for both lactosides and cellobiosides, and a marginally lower stability. However, kcat values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p–Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion.
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