Chirality-directed stem-cell-fate determination involves coordinated transcriptional and metabolomics programming that is only partially understood. Here, using high-throughput transcriptional-metabolic profiling and pipeline network analysis, the molecular architecture of chirality-guided mesenchymal stem cell lineage diversification is revealed. A total of 4769 genes and 250 metabolites are identified that are significantly biased by the biomimetic chiral extracellular microenvironment (ECM). Chirality-dependent energetic metabolism analysis has revealed that glycolysis is preferred during left-handed ECM-facilitated osteogenic differentiation, whereas oxidative phosphorylation is favored during right-handed ECM-promoted adipogenic differentiation. Stereo-specificity in the global metabolite landscape is also demonstrated, in which amino acids are enriched in left-handed ECM, while ether lipids and nucleotides are enriched in right-handed ECM. Furthermore, chirality-ordered transcriptomic-metabolic regulatory networks are established, which address the role of positive feedback loops between key genes and central metabolites in driving lineage diversification. The highly integrated genotype-phenotype picture of stereochemical selectivity would provide the fundamental principle of regenerative material design.

Late sodium current (I Na) inhibitors are a new subclass of antiarrhythmic agents. To overcome the drawbacks, e.g., low efficacy and inhibition effect on K+ current, of the FDA-approved late I Na inhibitor ranolazine, chain amide 6a–6q, 1,4-disubstituted piperazin-2-ones 7a–7s, and their derivatives 8a–8n were successively designed, synthesized, and evaluated in vitro on the NaV1.5-transfected HEK293T cells by the whole-cell patch clamp recording assay at the concentration of 40 μM. Among the new skeleton compounds, 7d showed the highest efficacy (IC50 = 2.7 μM) and good selectivity (peak/late ratio >30 folds), as well as excellent pharmacokinetics properties in mice (T 1/2 of 3.5 h, F = 90%, 3 mg/kg, po). It exhibited low hERG inhibition and was able to reverse the ATX-II-induced augmentation of late I Na phenotype of LQT3 model in isolated rabbit hearts. These results suggest the application potentials of 7d in the treatments of arrhythmias related to the enhancement of late I Na.

Here we designed enantiomeric lipid-mimetic glutamic acid derivatives (L/D-UG) and investigated their self-assembled chiral nanostructures' interaction with the protein adsorption as well as the osteogenesis. It was found that L or D-UG molecules can self-assemble into vesicle bilayers and two-dimensional (2D) nanocrystals via a kinetic and thermodynamic control, respectively. These chiral vesicles and 2D crystals showed differentiated adsorption of proteins, determined by their curvature and chirality. Specifically, fibronectin constituted by L-amino acids adsorbed preferentially on L-UG 2D crystal in a semi-random pattern and L-2D nanocrystal show as the most effective structures to promote bone regeneration. The controlled vesicle and 2D crystal assemblies with different chirality and curvature helps to clarify their determine roles in protein adsorption and osteogenesis.

In this study, a series of 2-[p-fluorophenyl]-6-substituted-9H-purine analogues were designed and synthesized as CHK1 inhibitors, among which compound b22 was the most potent. b22 exhibited nearly no antiproliferative activity toward HT29 cells and displayed a significant antitumor potentiating effect on HT29 cells when treated in combination with gemcitabine (Gem). A time-dependent assay found that treatment with Gem for 8 h before adding b22 achieved the optimal effect. Furthermore, the immunofluorescence and qPCR results demonstrated that b22 can remarkably reverse the upregulation of PD-L1 induced by Gem, which suggested dual effects of b22 in antitumor potentiation and antitumor immunity.

Inhibition of RNA-dependent RNA polymerase (RdRp) by nucleotide analogues with ribose modification provides a promising antiviral strategy for the treatment of SARS-CoV-2. Previous works have shown that remdesivir carrying 1'-substitution can act as a "delayed chain terminator", while nucleotide analogues with 2'-methyl group substitution could immediately terminate the chain extension. However, how the inhibition can be established by the 3'-ribose modification as well as other 2'-ribose modifications is not fully understood. Herein, we have evaluated the potential of several adenosine analogues with 2'- and/or 3'-modifications as obligate chain terminators by comprehensive structural analysis based on extensive molecular dynamics simulations. Our results suggest that 2'-modification couples with the protein environment to affect the structural stability, while 3'-hydrogen substitution inherently exerts "immediate termination" without compromising the structural stability in the active site. Our study provides an alternative promising modification scheme to orientate the further optimization of obligate terminators for SARS-CoV-2 RdRp.

In this study, a series of 2-substituted thieno[3,2-d]pyrimidin-4-yl(3,4,5-trimethoxyphenyl)methanones were designed, synthesized and evaluated as novel anti-tubulin polymerization and vascular disrupting agents. A pyrrolidin-1-yl derivative, compound 20, exhibited strong antiproliferative activities (average IC50 = 13.4 nM) against four cancer cell lines. 20 also showed retained potency toward paclitaxel-resistant A549 cells. 20 could significantly inhibit tubulin polymerization with an IC50 of 1.6 μM. 20 displayed strong induction of G2/M arrest and apoptosis through the mitochondrial pathway. Dose-dependent suppression of the migration of cancer cells and the formation of a vascular network were observed after treatment with 20. The acceptable microsomal stability implied that it is worth conducting further study on the analogues of 20 as novel drug candidates of CBSIs.

Remdesivir is one nucleotide analogue prodrug capable to terminate RNA synthesis in SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by two distinct mechanisms. Although the "delayed chain termination" mechanism has been extensively investigated, the "template-dependent" inhibitory mechanism remains elusive. In this study, we have demonstrated that remdesivir embedded in the template strand seldom directly disrupted the complementary NTP incorporation at the active site. Instead, the translocation of remdesivir from the +2 to the +1 site was hindered due to the steric clash with V557. Moreover, we have elucidated the molecular mechanism characterizing the drug resistance upon V557L mutation. Overall, our studies have provided valuable insight into the "template-dependent" inhibitory mechanism exerted by remdesivir on SARS-CoV-2 RdRp and paved venues for an alternative antiviral strategy for the COVID-19 pandemic. As the "template-dependent" inhibition occurs across diverse viral RdRps, our findings may also shed light on a common acting mechanism of inhibitors.

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1 '-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3 '-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1 '-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.

Regarding the nonclassical antifolates 6-(4'-methylphenethyl)-5-chloroacetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine (wm-8.2) as the lead compound, in order to improve molecular flexibility and simplify molecular structure, two series of compounds were designed and synthesized according to the 6-H and 6-methyl group. And the effects of carbon chain length and aromatic heterocyclic side chain on antitumor activity were investgated. Besides, the activities of key intermediates with molecular skeleton of folic acid inhibitor were measured to study the effect of the chloroacetyl group at N(5) position. Structures of 36 target compounds and key intermediates were confirmed by H-1 NMR, C-13 NMR and MS. The biological activity results showed that 6-methyl-2,4-diamino-5-(N-(4-methylphenyl)propyl-N-(2-chloroacetyl))aminopyrimidine (6b-3), which had three-carbon bridge and p-methylbenzene ring side chain, exhibited the best inhibition activities against HL-60, A549 and HCT116 cells with IC50 values as 0.25, 0.83 and 0.63 mu mol.L-1 respectively. 6-Methyl-2,4-diamino-5-(N-(4-methylphenyl)propyl)aminopyrimidine (5b-3), the key intermediate of 6b-3, showed excellent dihydrofolate reductase inhibitory activity. Molecular docking studies further explored the structure-activity relationship and possible causes of the difference inhibitory activity against dihydrofolate reductase.

A novel class of styryl sulfones were designed and synthesized as CAPE derivatives by our work team, which showed a multi-target neuroprotective effect, including antioxidative and anti-neuroinflammatory properties. However, the underlying mechanisms remain unclear. In the present study, the anti-Parkinson's disease (PD) activity of 10 novel styryl sulfone compounds was screened by the cell viability test and the NO inhibition test in vitro. It was found that 4d exhibited the highest activity against PD among them. In a MPTP-induced mouse model of PD, the biological activity of 4d was validated through suppressing dopamine neurotoxicity, microglial activation, and astrocytes activation. With compound 4d, we conducted the mechanistic studies about anti-inflammatory responses through inhibition of p38 phosphorylation to protect dopaminergic neurons, and antioxidant effects through promoting nuclear factor erythroid 2-related factor 2 (Nrf2). The results revealed that 4d could significantly inhibit 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP+)-induced p38 mitogen-activated protein kinase (MAPK) activation in both in vitro and in vivo PD models, thus inhibiting the NF-κB-mediated neuroinflammation-related apoptosis pathway. Simultaneously, it could promote Nrf2 nuclear transfer, and upregulate the expression of antioxidant phase II detoxification enzymes HO-1 and GCLC, and then reduce oxidative damage.

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N-5-electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N-5-chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC50 value of 12.1 mu M against MetH and 0.16-6.12 mu M against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G(1)-phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship. (C) 2020 Elsevier Masson SAS. All rights reserved.

A novel class of (E)-hydroxystyryl aralkyl sulfones were designed and synthesized as neuroprotective agents. Their neuroprotective properties were assessed by several antioxidant models including 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals scavenging model, neuronal protective effects against neurotoxins such as hydrogen peroxide (H2O2), 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium iodide (MPP+). Besides, the anti-inflammatory activity was evaluated by lipopolysaccharide (LPS)-induced nitric oxide (NO) release model in BV2 microglial cells. The result demonstrated that cinnamyl substituted compound5 hexhibited prominent antioxidant activity in H(2)O(2)and 6-OHDA models and higher anti-inflammatory potency (IC50=7.3 mu M) than lead compound1(IC50=13.4 mu M). Furthermore, compound5 hdisplayed predicted CNS (+) blood-brain barrier permeability (P-e=5.66x10(-6) cm s(-1)) in PAMPA model and low toxicity in PC12 and BV2 cells. These multifunctional properties highlight compound5 his a promising candidate for further development against neurodegenerative diseases.

: Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3 (cid:48) -5 (cid:48) and 2 (cid:48) -5 (cid:48) phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2 (cid:48) 3 (cid:48) -CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding a ffi nities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (K D = 0.038 µ M), a novel CDN that showed higher binding a ffi nity with hSTING than cGAMP (K D = 0.543 µ M). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists. β in human leukemic monocytes THP-1 by enzyme-linked immunosorbent assay (ELISA). The most promising analog, 8d was further evaluated for the ability to serve in other cytokine productions by RT-qPCR. We also explored the resistance of these compounds to cleavage by serum and nuclease P1 (NP1). We summarize our findings and discuss the structure-activity relationship observations interactions, which suggests that phosphodiester is an active binding site. molecular our These provide guidelines targeted STING. assay as aptamers, antisense and adjuvant candidates for improving immunotherapy potency against and Large-scale drug modification and agonists on

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3′-5′ and 2′-5′ phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2′3′-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (KD = 0.038 μM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (KD = 0.543 μM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.

In the present study,novel ester derivatives of CAPE were designed and synthesized as neuroprotective agents.The anti-inflammatory and antioxidant activities of these compounds were evaluated at the cellular level,while the blood-brain barrier (BBB) permeability was predicted by parallel artificial membrane permeability assay (PAMPA).The results revealed that phenolic hydroxyl groups and double bonds in the structure of CAPE had important effects on neuroprotective activities.Accordingly,a preliminary structure-activity relationship was summarized in this paper.In addition,we observed a significant improvement on BBB permeability.These results provided important references for the structural modification and optimization of CAPE in the future.

Using compounds 2,4-diamino-6-(4-methylphenethyl) pyrido [3,2-d] pyrimidine(wm-5b) and 2,4-diaminopyrido [3,2-d] pyrimidine as the lead compounds, a series of 2- or 4-substituted pyrido [3,2-d] pyrimidines was synthesized as potential nonclassical antifolates by means of microwave efficiently. Besides, the effects of different substituted positions which provides more theoretical basis for the design and synthesis of nonclassical antifolates can be explored. The target compounds were characterized by H-1 NMR, C-13 NMR as well as MS. All compounds showed certain anti-tumor activity [compound 6b, the most potent one, has IC50 values of (4.09 +/- 0.48) mu mol/L against HL-60 cells, IC50 value of (17.99 +/- 7.20) mu mol/L against A549 cells and IC50 value of (14.52 +/- 4.74) mu mol/L against HCT116 cells], while some of them exhibited certain inhibition of recombinant human DHFR(rhDHFR). Some compounds and the lead were applied to molecular docking of the crystal structure of dihydrofolate reductase, and the activity results and structure-activity relationship were explained at the molecular level.

Summary of main observation and conclusionThe SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic fragment, proteolytic recognition sites (such as DDDDK, AVLQ, and WELQ) were directly engineered into the first or second loop of SpyCatcher at locations after the reactive lysine to give a set of cleavable SpyCatcher variants. Among them, SpyCatcherDDDDK exhibits excellent reactivity with SpyTag and could still be cleaved proteolytically by enterokinase after ligation. Notably, SpyCatcherDDDDK is disordered in solution and forms an ordered complex upon reaction with SpyTag with a second order rate constant of 99.2 ± 0.1 M–1·s–1, which is comparable to, if not faster than, most click reactions. The results demonstrate the high sequence plasticity of SpyCatcher and suggest that covalent bond formation may confer robustness on the folded structure against extensive mutation. These variants add to the expanding toolbox of genetically‐encoded peptide‐protein chemistry with diverse features.

(E)-3,4-dihydroxystyryl alkyl sulfones, as new analogues of neurodegenerative agents, were designed and synthesized. The biological results demonstrated that most of the target compounds preserved antioxidant and anti-inflammatory potency in scavenging reactive free radicals, protecting neuronal cells against neurotoxins such as H2O2, 6-hydroxydopamine and inhibiting lipopolysaccharide (LPS)-induced over-production of NO. Among these compounds, 6.22 with cyclopentyl propyl exhibited prominent antioxidant activity at low concentration (2.5 μM) in H2O2 model (cell viability = 94.5%). In addition, 6.22 (IC50 = 1.6 μM) displayed better anti-inflammatory activity than that of lead compound 1 (IC50 = 13.4 μM). In view of the outstanding performance of 6.22, the apoptotic rates of H2O2-damaged PC12 cells were detected by Annexin V-FITC/PI assay. 6.22 showed higher potency in inhibition of apoptosis than 1 at low concentration (2.5 μM), consisting with the antioxidant and anti-inflammatory models. Furthermore, with the predicted CNS (+) blood-brain barrier (BBB) permeability (Pe = 6.84 × 10-6 cm s-1), low cytotoxicity and favorable physiochemical properties based on calculation, compound 6.22 can be further developed as a potential multifunctional neuroprotective agent.

The emergence of "molecular superglue," such as SpyTag-SpyCatcher chemistry, has tremendously expanded our capability in manipulating protein shape and architecture via conjugation. Telechelic proteins bearing the SpyTag and SpyCatcher reactive sequences can be expressed and purified for bioconjugation in vitro, giving protein conjugates, branched proteins, and circular proteins. By encoding both reactive sequences in the same construct for expression in vivo, the nascent protein undergoes programmed posttranslational modification guided by protein folding and reaction, leading to diverse nonlinear topologies in situ. In this chapter, we present the SpyTag-SpyCatcher chemistry as a versatile platformfor protein bioconjugation and topology engineering.