Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ...ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP K m of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or K m ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than K m ATP may provide crucial insights into a compound’s activity and selectivity in cellular contexts.
We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. ...Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an ...attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.
Pharmacokinetic properties of our first-generation HIF-2α antagonist PT2385, including modest solubility, resulted in a high recommended phase 2 dose (RP2D) of 800 mg BID and motivated the pursuit of ...novel scaffolds which could improve solubility and formulation parameters with the goal of improved pharmacokinetics. Herein we disclose our successful efforts to identify such HIF-2α antagonists through an optimization strategy characterized by: (1) increasing the fraction of sp
3
hybridized carbons (Fsp
3
), (2) replacing the aromatic portion of the indane core with pyridine heterocycles, and (3) improving a putative O
lp
→π*
Ar
interaction, an underutilized electrostatic contact in medicinal chemistry. These efforts emphasize the importance of employing multiple strategies in parameter optimization. In isolation, modifications to areas (1) and (2) improved solubility, but with the compromise of reduced potency. In area (3), understanding the importance of an O
lp
→π*
Ar
interaction, as documented through a wealth of crystal structures and retrospective calculations, proved essential in guiding SAR and identifying the trifluoromethyl group as a suitable replacement of the sulfone. Only by combining these three strategies could inhibitors with substantially improved solubility and comparable potency be discovered. Finally, the overall improvement in pharmacokinetic properties of the newly identified inhibitors is highlighted through a battery of ADME and in vivo data, including use of pharmacodynamic biomarkers indicative of HIF-2α antagonism.
One-time password algorithms are widely used in digital services to improve security. However, many such solutions use a constant secret key to encrypt (process) one-time plaintexts. A paradigm shift ...from constant to one-time keys could introduce tangible benefits to the application security field. This paper analyzes a one-time password concept for the Rivest–Shamir–Adleman algorithm, in which each key element is hidden, and the value of the modulus is changed after each encryption attempt. The difference between successive moduli is exchanged between communication sides via an unsecure channel. Analysis shows that such an approach is not secure. Moreover, determining the one-time password element (Rivest–Shamir–Adleman modulus) can be straightforward. A countermeasure for the analyzed algorithm is proposed.
Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by ...several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X‐ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the β‐anomer of the substrate. A triad of polar residues in the second‐shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.
Acidic mammalian chitinase (AMCase) is a member of the glycosyl hydrolase 18 family (EC 3.2.1.14) that has been implicated in the pathophysiology of allergic airway disease such as asthma. Small ...molecule inhibitors of AMCase were identified using a combination of high-throughput screening, fragment screening, and virtual screening techniques and characterized by enzyme inhibition and NMR and Biacore binding experiments. X-ray structures of the inhibitors in complex with AMCase revealed that the larger more potent HTS hits, e.g. 5-(4-(2-(4-bromophenoxy)ethyl)piperazine-1-yl)-1H-1,2,4-triazol-3-amine 1, spanned from the active site pocket to a hydrophobic pocket. Smaller fragments identified by FBS occupy both these pockets independently and suggest potential strategies for linking fragments. Compound 1 is a 200 nM AMCase inhibitor which reduced AMCase enzymatic activity in the bronchoalveolar lavage fluid in allergen-challenged mice after oral dosing.
Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and ...allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3−6, human AMCase has only one pH optimum for k cat/K m between pH 4 and 5. Steady state kinetics shows that human AMCase has “low” intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4−6-mers) suggests that human AMCase prefers the β anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.
Designs of timer function blocks (FBs) are presented in the article. The developed modules are IEC 61131-3. An analysis of IEC 61131-3 in terms of timer functionality and implementation options is ...presented. Three types are presented, timer-on, timer-off, and timer-pulse, with each type designed to be fully hardware or software-like. Both designs, hardware or software-like, can operate as multi-channel timers. Particularly noteworthy is the software-like design, for which a solution without edge detectors was achieved. Such a feature was obtained by reversing the method of time determination by counting the difference between the start and end times and by using specific features of the D flip-flops, that is, clock-enable inputs. The presented timers were written in Verilog language and implemented in an FPGA chip. Thanks to the universal design of the interface, the proposed FBs can be used for the hardware support of existing programmable logic controllers (PLCs) or as an integral part of newly built PLC CPUs. The idea of a CPU architecture with hardware support is proposed. The paper presents the results of the implementation in an FPGA of the Kintex UltraScale+ family from AMD-Xilinx.
This article discusses edge detectors implemented in programmable logic controllers. The behaviors of different vendors’ solutions are presented with pros and cons. The trigger functions defined in ...the IEC 61131-3 standard were analyzed for implementations. The main contribution of this paper is an idea for hardware acceleration of standard trigger functions that enables us to build single-clock-cycle edge detectors. Additionally, the structure for automatic edge detection on every input is shown. The structure with a synthesizable Verilog HDL description is presented. The comparison of the solution with vendor programmable logic controllers (PLCs) proves the effectiveness of the designed hardware-aided unit.
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