Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif

The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment (¹⁸⁵QP P VP P QRPM¹⁹⁴, termed as SLP76^185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76^185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76^185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76^185–194(N‐Leu187/N‐Gln190) and SLP76^185–194(N‐Thr187/N‐Gln190), are designed to have high potency (K_d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide (K_d = 5.7 ± 1.2 μM).     

Sex‐specific behavioral traits in the Brd2 mouse model of juvenile myoclonic epilepsy

Idiopathic generalized epilepsy represents about 30–35% of all epilepsies in humans. The bromodomain BRD2 gene has been repeatedly associated with the subsyndrome of juvenile myoclonic epilepsy (JME). Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/?) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased gamma‐aminobutyric acid (GABA) markers in the direct basal ganglia pathway as well as in the neocortex and superior colliculus. Here, we tested male and female Brd2+/? and wild‐type littermate mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) to identify whether Brd2 haploinsufficiency is associated with the human behavioral patterns, the so‐called JME personality. Brd2+/? females but not males consistently displayed decreased anxiety. Furthermore, we found a highly significant dominance trait (aggression) in the Brd2+/? mice compared with the wild type, more pronounced in females. Brd2+/? mice of either sex did not differ from wild‐type mice in spatial learning and memory tests. Compared with wild‐type littermates, we found decreased numbers of GABA neurons in the basolateral amygdala, which is consistent with the increase in aggressive behavior. Our results indicate that Brd2+/? haploinsufficient mice show no cognitive impairment but have behavioral traits similar to those found in patients with JME (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of JME or it controls upstream regulators of individual phenotypes.     

Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking

A profoundly time‐efficient chemoenzymatic method for the synthesis of (S)‐3‐(4‐chlorophenoxy)propan‐1,2‐diol and (S)‐1‐chloro‐3‐(2,5‐dichlorophenoxy)propan‐2‐ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R‐ and S‐enantiomers of the intermediates demonstrated stronger H‐bond interaction between the hydroxyl group of the R‐enantiomer and the key binding residues of the catalytic site of the lipase, while the S‐enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost‐effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.     

RAC1mutation is not a predictive biomarker for PI3’‐kinase‐β‐selective pathway‐targeted therapy

Mutational activation of RAC1 is detected in ~7% of cutaneous melanoma, with the most frequent mutation (RAC1^C85T) encoding for RAC1^P29S. RAC1^P29S is a fast‐cycling GTPase that leads to accumulation of RAC1^P29S‐GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1^P29S propagates its pro‐tumorigenic effects remains unclear. RAC1‐GTP is reported to activate the beta isoform of PI3’‐kinase (PIK3CB/PI3Kβ) leading to downstream activation of PI3’‐lipid signaling. Hence, we employed both genetic and isoform‐selective pharmacological inhibitors to test if RAC1^P29S propagates its oncogenic signaling in melanoma through PI3Kβ. We observed that RAC1^P29S‐expressing melanoma cells were largely insensitive to inhibitors of PI3Kβ. Furthermore, RAC1^P29S melanoma cell lines showed variable sensitivity to pan‐class 1 (α/β/γ/δ) PI3’‐kinase inhibitors, suggesting that RAC1‐mutated melanoma cells may not rely on PI3’‐lipid signaling for their proliferation. Lastly, we observed that RAC1^P29S‐expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 → PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1‐mutated melanoma.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

Solid Phase Synthesis of Phosphopeptides Incorporating 2,2-Dimethyloxazolidine Pseudoproline Analogs: Evidence for <Emphasis Type="Italic">trans</Emphasis> Leu-Pro Peptide Bonds in Stat3 Inhibitors

To answer the question of whether the conformation of the Leu-Pro bond is cis or trans in Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH₂ when complexed with the SH2 domain of Stat3, we substituted 2,2-dimethyloxazolidines derived from serine (Ser(Ψ^Me,Mepro)) and threonine (Thr(Ψ^Me,Mepro)) for proline. The 2,2-dimethyloxazolidine and 2,2-dimethylthiazolidine pseudoproline (ΨPro) analogs induce predominantly cis Xxx-ΨPro peptide bonds. As these ΨPro analogs are acid-labile, the phosphopeptides were synthesized using Fmoc-based SPPS using unprotected phosphotyrosine and 4-hydroxybenzoate as the linker that allowed release from the support by alkaline ammonolysis, conditions that kept the oxazolidine rings intact. Incorporation of Ser(Ψ^Me,Mepro) resulted in 69% cis Leu-ΨPro bond content in aqueous solution whereas that for Thr(Ψ^Me,Mepro) analog was 63%. Affinities for Stat3 were 3–5 fold lower than the lead compound and were inversely correlated with cis content. Thus we conclude that the Leu-Pro peptide bond is trans when the peptide is bound to Stat3.     

Circularly polarized luminescence of Sm (III) and Eu (III) complexes with chiral ligand (R/S)‐BINAPO

Luminescent lanthanide (III) ions have been exploited for circularly polarized luminescence (CPL) for decades. However, very few of these studies have involved chiral samarium (III) complexes. Complexes are prepared by mixing axial chiral ligands (R/S))‐2,2’‐bis(diphenylphosphoryl)‐1,1′‐binaphthyl (BINAPO) with europium and samarium Tris (trifluoromethane sulfonate) (Eu (OTf)₃ and Sm (OTf)₃). Luminescence‐based titration shows that the complex formed is Ln((R/S)‐BINAPO)₂(OTf)₃, where Ln = Eu or Sm. The CPL spectra are reported for Eu((R/S)‐BINAPO)₂(OTf)₃ and Sm((R/S)‐BINAPO)₂(OTf)₃. The sign of the dissymmetry factors, g_em, was dependent upon the chirality of the BINAPO ligand, and the magnitudes were relatively large. Of all of the complexes in this study, Sm((S)‐BINAPO)₂(OTf)₃ has the largest g_em = 0.272, which is one of the largest recorded for a chiral Sm³⁺ complex. A theoretical three‐dimensional structural model of the complex that is consistent with the experimental observations is developed and refined. This report also shows that (R/S)‐BINAPO are the only reported ligands where g_em (Sm³⁺) > g_em (Eu³⁺).     

Genetic variation in productivity of foundation riparian species at the edge of their distribution: implications for restoration and assisted migration in a warming climate

We examined the hypothesis that genotypic variation among populations of commonly co‐occurring phreatophytic trees (Populus fremontii, Salix gooddingii) and the shrub (Salix exigua) regulates aboveground net primary productivity (ANPP) at a hot site at the edge of the species’ distribution. We used a provenance trial in which replicated genotypes from populations varying in mean annual temperature were transplanted to a common garden adjacent to the Lower Colorado River in southeastern California. The garden environment represented an extreme maximum temperature for the study species. Four major findings emerged: (1) Genotypic variation in ANPP was significant for all species with broad‐sense heritability (H²) across populations of 0.11, 0.13, and 0.10 for P. fremontii, S. gooddingii, and S. exigua, respectively, and within‐population H² ranging from 0.00 to 0.25, 0.00 to 0.44, and 0.02 to 0.21, respectively. (2) Population ANPP decreased linearly as mean annual maximum temperature (MAMT) transfer distance increased for both P. fremontii (r² = 0.64) and S. gooddingii (r² = 0.37), whereas it did not change for S. exigua; (3) Populations with similar MAMT to that of the common garden were 1.5 and 1.2 times more productive than populations with 5.0 °C MAMT transfer distances for P. fremontii and S. gooddingii, respectively; and (4) Variation in regression slopes among species for the relationship between ANPP and MAMT indicate species‐specific responses to temperature. As these plant species characterize a threatened habitat type and support a diverse community that includes endangered species, ecosystem restoration programs should consider using both local genotypes and productive genotypes from warmer environments to maximize productivity of riparian ecosystems in the face of global climate change.     

Antimicrobial Activity of Monoketone Curcuminoids Against Cariogenic Bacteria

We evaluated the antimicrobial activity of 25 monoketone curcuminoids (MKCs) against a representative panel of cariogenic bacteria in terms of their minimum inhibitory concentration (MIC) values. Curcumin A ( 10 ) displayed promising activity against Streptococcus mutans (MIC = 50 μg/ml) and Streptococcus mitis (MIC = 50 μg/ml) as well as moderate activity against S. sanguinis (MIC = 100 μg/ml), Lactobacillus casei (MIC = 100 μg/ml), and Streptococcus salivarius (MIC = 200 μg/ml). Results indicated higher activity of compound 10 than that of its bis‐β‐diketone analog. Additionally, compounds 3a (1,5‐bis(4‐methylphenyl)pentan‐3‐one) and 7b (1,5‐bis(4‐bromophenyl)pentan‐3‐ol) were moderately active against S. mitis (MIC = 100 μg/ml) and S. salivarus (MIC = 200 μg/ml).     

Design,synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/T790M/C797S mutation

The efficacy of EGFR inhibitors is frequently affected by acquired resistance. EGFR^{19D/T790M/C797S} mutation is one of the primary reasons for the emergence of resistance after treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib. To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and docking analysis were also carried out. Among them, compound 12e displayed significant inhibitory activity against EGFR^{19D/T790M/C797S} (IC₅₀ = 15.3 nM) and good selectivity over EGFR WT (IC₅₀ > 1000 nM), L858R/T790M (IC₅₀, 156.6 nM) and L858R/T790M/C797S (IC₅₀, 218.3 nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase cell cycle arrest and apoptosis in BaF3/EGFR^{19D/T790M/C797S} cells by suppressing EGFR phosphorylation signaling pathway. In all, our study might provide a novel structural design method and lay the solid foundation for the development of the 4th generation EGFR^{19D/T790M/C797S} inhibitors.     

The role of environmental, root, and microbial biomass characteristics in soil respiration in temperate secondary forests of Northeast China

For secondary forests, the major forest resources in China (accounting for more than 50% of the national total), soil respiration (R _S) and the relationship between R _S and various biotic/abiotic factors are poorly understood. The objectives of the present study were to examine seasonal variations in soil respiration during the growing season, and to explore the factors affecting the variation in soil respiration rates for three forest types (Mongolian oak, Manchurian walnut and mixed forests) of temperate secondary forest in Northeast China. The results showed that (1) the maximum total R _S rate occurred in July, following a bell-shaped curve with season, (2) for all forest types, the total R _S was significantly influenced by soil temperature (P < 0.01), and did not significantly correlate with soil moisture, (3) compared with fine root biomass, coarse root biomass was more closely related with the root respiration in mixed forest (R ² = 0.711, P = 0.017) and in Manchurian walnut forest (R ² = 0.768, P = 0.010), and (4) microbial biomass carbon (MBC) and nitrogen were significantly correlated with heterotrophic R _S in Mongolian oak forest (R ² = 0.664, P = 0.026; R ² = 0.784, P = 0.008, respectively) and in mixed forest (R ² = 0.918, P = 0.001; R ² = 0.967, P = 0.001, respectively). We can conclude that in temperate secondary forests: (1) the R _S rate and the relationships between R _S and abiotic/biotic factors change greatly with forest types, and (2) R _S is strongly influenced by soil temperature, MBC, microbial biomass nitrogen and coarse root biomass in temperate secondary forests.     

Brucin,an antibacterial peptide derived from fruit protein of Fructus Bruceae,Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l^?1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

Significance and Impact of the Study

An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L.) Merr. by pepsin‐catalysed hydrolysis. Its inhibitory activity towards the Gram‐positive bacteria was higher than penicillin G and chloramphenicol. The result suggested that Brucin may be applied for the treatment of the disease caused by Strep. pyogenes^*.     

Brd4‐Brd2 isoform switching coordinates pluripotent exit and Smad2‐dependent lineage specification

Pluripotent stem cells (PSCs) hold great clinical potential, as they possess the capacity to differentiate into fully specialised tissues such as pancreas, liver, neurons and cardiac muscle. However, the molecular mechanisms that coordinate pluripotent exit with lineage specification remain poorly understood. To address this question, we perform a small molecule screen to systematically identify novel regulators of the Smad2 signalling network, a key determinant of PSC fate. We reveal an essential function for BET family bromodomain proteins in Smad2 activation, distinct from the role of Brd4 in pluripotency maintenance. Mechanistically, BET proteins specifically engage Nodal gene regulatory elements (NREs) to promote Nodal signalling and Smad2 developmental responses. In pluripotent cells, Brd2‐Brd4 occupy NREs, but only Brd4 is required for pluripotency gene expression. Brd4 downregulation facilitates pluripotent exit and drives enhanced Brd2 NRE occupancy, thereby unveiling a specific function for Brd2 in differentiative Nodal‐Smad2 signalling. Therefore, distinct BET functionalities and Brd4‐Brd2 isoform switching at NREs coordinate pluripotent exit with lineage specification.     

A highly selective and sensitive turn‐on fluorescent probe for the detection of Al3+ and its bioimaging

A novel fluorescent sensor, 1‐((2‐hydroxynaphthalen‐1‐yl)methylene)urea (ocn) has been designed and applied as a highly selective and sensitive fluorescent probe for recognition of Al³⁺ in Tris–HCl (pH = 7.20) solution. The probe ocn exhibits an excellent selectivity to Al³⁺ over other examined metal ions, anions and amino acids with a prominent fluorescence ‘turn‐on’ at 438 nm. ocn binds to Al³⁺ with a 2:1 binding stoichiometry and the detection limit was 0.3 μM. Furthermore, its capability of biological application was evaluated and the results showed that the sensor could be used to detect Al³⁺ in living cells.