Novel Inhibitor Discovery through Virtual Screening against Multiple Protein Conformations Generated via Ligand-Directed Modeling: A Maternal Embryonic Leucine Zipper Kinase Example

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein–ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (<b>15</b>; K_d = 0.37 μM), one moderately active (<b>12</b>; K_d = 3.2 μM), and one weak but very selective (<b>9</b>; K_d = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general

Switchable Adhesive Based on Shape Memory Polymer with Micropillars of Different Heights for Laser-Driven Noncontact Transfer Printing

Switchable adhesive is essential to develop transfer printing, which is an advanced heterogeneous material integration technique for developing electronic systems. Designing a switchable adhesive with strong adhesion strength that can also be easily eliminated to enable noncontact transfer printing still remains a challenge. Here, we report a simple yet robust design of switchable adhesive based on a thermally responsive shape memory polymer with micropillars of different heights. The adhesive takes advantage of the shape-fixing property of shape memory polymer to provide strong adhesion for a reliable pick-up and the various levels of shape recovery of micropillars under laser heating to eliminate the adhesion for robust printing in a noncontact way. Systematic experimental and numerical studies reveal the adhesion switch mechanism and provide insights into the design of switchable adhesives. This switchable adhesive design provides a good solution to develop laser-driven noncontact transfer printing with the capability of eliminating the influence of receivers on the performance of transfer printing. Demonstrations of transfer printing of silicon wafers, microscale Si platelets, and micro light emitting diode (μ-LED) chips onto various challenging nonadhesive receivers (e.g., sandpaper, stainless steel bead, leaf, or glass) to form desired two-dimensional or three-dimensional layouts illustrate its great potential in deterministic assembly

Switchable Adhesive Based on Shape Memory Polymer with Micropillars of Different Heights for Laser-Driven Noncontact Transfer Printing

Switchable adhesive is essential to develop transfer printing, which is an advanced heterogeneous material integration technique for developing electronic systems. Designing a switchable adhesive with strong adhesion strength that can also be easily eliminated to enable noncontact transfer printing still remains a challenge. Here, we report a simple yet robust design of switchable adhesive based on a thermally responsive shape memory polymer with micropillars of different heights. The adhesive takes advantage of the shape-fixing property of shape memory polymer to provide strong adhesion for a reliable pick-up and the various levels of shape recovery of micropillars under laser heating to eliminate the adhesion for robust printing in a noncontact way. Systematic experimental and numerical studies reveal the adhesion switch mechanism and provide insights into the design of switchable adhesives. This switchable adhesive design provides a good solution to develop laser-driven noncontact transfer printing with the capability of eliminating the influence of receivers on the performance of transfer printing. Demonstrations of transfer printing of silicon wafers, microscale Si platelets, and micro light emitting diode (μ-LED) chips onto various challenging nonadhesive receivers (e.g., sandpaper, stainless steel bead, leaf, or glass) to form desired two-dimensional or three-dimensional layouts illustrate its great potential in deterministic assembly

Switchable Adhesive Based on Shape Memory Polymer with Micropillars of Different Heights for Laser-Driven Noncontact Transfer Printing

Switchable adhesive is essential to develop transfer printing, which is an advanced heterogeneous material integration technique for developing electronic systems. Designing a switchable adhesive with strong adhesion strength that can also be easily eliminated to enable noncontact transfer printing still remains a challenge. Here, we report a simple yet robust design of switchable adhesive based on a thermally responsive shape memory polymer with micropillars of different heights. The adhesive takes advantage of the shape-fixing property of shape memory polymer to provide strong adhesion for a reliable pick-up and the various levels of shape recovery of micropillars under laser heating to eliminate the adhesion for robust printing in a noncontact way. Systematic experimental and numerical studies reveal the adhesion switch mechanism and provide insights into the design of switchable adhesives. This switchable adhesive design provides a good solution to develop laser-driven noncontact transfer printing with the capability of eliminating the influence of receivers on the performance of transfer printing. Demonstrations of transfer printing of silicon wafers, microscale Si platelets, and micro light emitting diode (μ-LED) chips onto various challenging nonadhesive receivers (e.g., sandpaper, stainless steel bead, leaf, or glass) to form desired two-dimensional or three-dimensional layouts illustrate its great potential in deterministic assembly

FRRFa modulates L280C after MTSET modification.

<p><b>A</b>. Schematic of experimental design and representative traces. Left Panel: L280C was activated with pH 5.0 with and without 100 µM FRRFa. Right Panel: L280C was modified with 300 µM MTSET for 3 minutes at pH 7.4. After treatment, MTSET was removed by washing with pH 7.4 and pH 5.0-evoked current was recorded in the absence of FRRFamide. Channels were then washed with pH 7.4 and allowed to recover for 1 minute. Then pH 7.4 solutions containing 100 µM FRRFa were applied for 1 minute. After application of FRRFa, channels were activated with pH 5.0. For quantification (<b>B</b>–<b>D</b>), % change was determined by subtracting the stated characteristic (peak amplitude, residual current, or τ_inact) with FRRFa from control (no peptide) and normalized to the no peptide response. <b>B</b>. Quantification of % change in peak current amplitude. The magnitude of the change in peak current amplitude evoked with FRRFa was independent of MTSET modification (<i>n</i> = 8-11, <i>p</i> = 0.6). <b>C</b>. Quantification of the % change in the rate of inactivation (τ_inact). FRRFa response on inactivation after MTSET was not significantly different from FRRFa response on unmodified L280C (<i>n</i> = 8-11, <i>p</i> = 0.9). <b>D</b>. Quantification of % change in residual current. After MTSET modification, FRRFa still increased residual current (<i>n</i> = 8, <i>p</i> = 0.02, paired Student’s t-test), but this was not as robust as FRRFa-induced residual current of unmodified L280C (<i>n</i> = 8-11, <i>p</i> = 0.03). Data are mean ± SEM. “*” indicates <i>p</i> < 0.05 and n.s. indicates no significant difference.</p

MTSET modification of L280C, I307C, and L415C.

<p><b>A</b>. Effect of MTSET on wildtype or mutant ASIC1a expressed in <i>Xenopus</i> oocytes. MTSET (300 µM) was applied at pH 7.4 for 3 minutes and removed by washing with pH 7.4 solutions. pH 5.0-evoked currents after MTSET incubation (“+ MTSET”) were compared to control currents in the same oocyte measured before MTSET application (“Control”). <b>B</b>–<b>C</b>. Quantification of (<b>B</b>) tau of inactivation (<i>n</i> = 6-8) and (<b>C</b>) residual current (<i>n</i> = 6-7). <b>D</b>. Representative recordings of steady-state desensitization (SSD) before and after MTSET modification. Oocytes were maintained at a basal of pH 7.9 and then incubated with pH 6.7 for 2 minutes to induce SSD (shaded bars) prior to activation with pH 5.0 (white bars). <b>E</b>. Quantification of MTSET-dependent changes in SSD (<i>n</i> = 5-6). <b>F</b>. Representative traces of MTSET exposure on pH-dependent activation. MTSET was applied as above and the response to pH 5.0 (white bars) or pH 6.5 (gray bars) from basal pH 7.4 was measured. <b>G</b>. Quantification of pH 6.5-mediated activation before and after MTSET modification (<i>n</i> = 6-8). Data are mean ± SEM. “**” and “***” indicate <i>p</i>-values < 0.01 and 0.001, respectively. Significance was determined with paired Student’s t-tests.</p

FRRFa modulation of L280C.

<p><b>A</b>. FRRFa increases residual current of wildtype and L280C. FRRFa (100 µM) was applied for 1 minute at pH 7.4 prior to activation with pH 5.0. For L280C, arrowheads highlight the FRRFa-induced increase in peak current amplitude. <b>B</b>. Quantification of residual current (<i>n</i> = 14-17). <b>C</b>. Quantification of FRRFa modulation on pH 5.0-evoked peak current amplitude. The percent change in amplitude was determined by subtracting the pH 5.0-evoked peak current amplitude of vehicle from the pH 5.0 evoked peak current amplitude evoked after FRRFamide modulation and normalizing to the vehicle peak current amplitude from the same cell (<i>n</i> = 17-25). <b>D</b>. Representative trace of FRRFa modulation on steady-state desensitization. 100 µM FRRFa or vehicle was applied for 1 minute at basal pH 7.4 and again during the 2 minute incubation with conditioning pH 6.7. Proton-gated current was evoked with pH 5.0 (white bar). <b>E</b>. Quantification of FRRFa modulation of steady-state desensitization (<i>n</i> = 4). <b>F</b>–<b>G</b>. FRRFamide concentration response curve for wildtype ASIC1a (<b>F</b>) and L280C (<b>G</b>). The effect of FRRFamide on residual current was assessed. Our data suggest that 100 µM FRRFa induced a maximal response on wildtype ASIC1a as 300 µM FRRFa was not significantly different from 100 µM FRRFa (<i>n</i> = 5, <i>p</i> = 0.69 paired Student’s t-test, difference between 100µM and 300µM was 10.42% ± 11.25%; <i>data not shown</i>). Based on this information, the calculated EC₅₀ for wildtype ASIC1a was 20 ± 4 µM and 14 ± 4 µM for L280C (<i>n</i> = 6-8, <i>p</i> = 0.34). Data are mean ± SEM. “**” and “***” indicates <i>p</i>-value < 0.01 and 0.001 respectively.</p

sj-docx-1-pie-10.1177_09544089221126779 - Supplemental material for Effect of interlayer rescanning on surface roughness and overhang sinking distance of AlSi10Mg alloy via selective laser melting

Supplemental material, sj-docx-1-pie-10.1177_09544089221126779 for Effect of interlayer rescanning on surface roughness and overhang sinking distance of AlSi10Mg alloy via selective laser melting by Yiqing Ma, Meiping Wu, Weipeng Duan, Xiaojie Shi, Huijun Liu, Chenglong Li and Xiaojin Miao in Proceedings of the Institution of Mechanical Engineers, Part E: Journal of Process Mechanical Engineering</p

Location and characteristics of L280C, I307C, and L415C in human ASIC1a.

<p><b>A</b>–<b>B</b>. Human ASIC1a was modeled based on the chicken ASIC1 crystal structure (PDB ID: 3HGC). One subunit has been removed to show the inside of the central vestibule. The subunits are color-coded to highlight different regions of the ASIC1a structure. The boxed region is magnified in <b>B</b> to illustrate the positions of L280, I307, and L415. Images were rendered using the UCSF Chimera package [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071733#B63" target="_blank">63</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071733#B64" target="_blank">64</a>]. <b>C</b>. Representative recordings of acid-activated currents in <i>Xenopus</i> oocytes expressing wild-type human ASIC1a, L280C, I307C, or L415C. Basal pH was maintained at pH 7.4 before application of pH 5.0 (white bars above trace). <b>D</b>. Quantification of the tau of inactivation (<i>n</i> = 10-14), calculated through a single exponential fit of the decay phase of the acid-evoked current. <b>E</b>. Quantification of proton-dependent activation (<i>n</i> = 6-9). I/I_max is the peak current amplitude evoked from test pH conditions normalized to peak current amplitude evoked with pH 5.0. <b>F</b>. Quantification of the proton-dependence of steady-state desensitization (<i>n</i> = 6-10). I/I_max. is the peak current amplitude of pH 5.0-evoked currents after conditioning in the test pH normalized to the pH 5.0-evoked current after conditioning in pH 7.9 (see methods). Data are mean ± SEM. “***” indicates a <i>p</i>-value < 0.001, respectively. “n.s.” indicates no significant difference.</p

FRRFa impairs MTSET modification.

<p><b>A</b>. Schematic of MTSET/peptide experimental design. 100 µM peptide or vehicle was applied for 1 minute before application of MTSET (in the presence of vehicle or peptide). Excess MTSET was removed by washing in peptide (or vehicle) containing solutions. Finally, peptide (or vehicle) was washed-out thoroughly and residual current was measured. <b>B</b>. Representative traces of L280C pH 5.0-evoked currents following 2.5 minutes of MTSET modification in the presence of vehicle, FRRFa, or FRRF (no amide). <b>C</b>. Quantification of residual current after exposure to MTSET in the presence of vehicle, 100 µM FRRFa, or 100 µM FRRF (no amide). Curves represent one-phase association exponential fits of the data points. FRRFa significantly altered MTSET modification (<i>p</i> < 0.001, 2-way ANOVA). No difference was observed between Vehicle and FRRF (no amide). Data are mean ± SEM, <i>n</i> = 3-22.</p