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Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the α-kinase family that is activated by calcium/calmodulin. Of note, eEF2K is crucial for regulating translation and is often highly overexpressed in malignant cells. Therefore in this review, we summarize the molecular structure of eEF2K and its oncogenic roles in cancer. Moreover, we further discuss the inhibition of eEF2K with small-molecule inhibitors and other new emerging therapeutic strategies in cancer therapy. Taken together, these inspiring findings provide new insights into a promising strategy for inhibiting eEF2K to greatly improve future cancer therapy.

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Resin mineral composite (RMC) is a new material with several times the damping properties of gray cast iron and great corrosion resistance. Due to its overall brittleness, sawing with a diamond band saw would be a suitable method. In this research, sawing experiments are carried out to study the sawing force characteristics of the material and its surface morphology during the processing. The results show that the feed force level is in the range of 3.5~5.5 N and the tangential force level is relatively low. The distribution of resin mineral components does not have a significant impact on the average sawing force but increases the fluctuation of the lateral force signal. The maximum fluctuation volume is 94.86% higher than other areas. Uneven lateral force, generated when diamond particles pass through the resin-mineral interface, is one of the causes of fluctuations. The machined surface of RMC has uniform strip scratches and a small number of pits. Maintaining a constant ratio of sawing speed to feed speed can result in approximately the same machined surface. A step structure with a height of about 10 µm appears at the interface of resin minerals. As a processing defect, it may affect the performance of RMC components in some aspects, which need a further precision machining processing.

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In recent years, PD-1 immune checkpoint inhibitors based on monoclonal antibodies have revolutionized cancer therapy, but there still exist unresolved issues, such as the high cost, the relatively low response rates, and so on, compared with small-molecule drugs. Herein a type of pyrrole-imidazole (Py-Im) polyamide as a small-molecule DNA binder was designed and synthesized, which could competitively bind to the same double-stranded DNA stretch in the PD-L1 promoter region as the STAT3 binding site and thus downregulate PD-L1 expression. It was demonstrated that the Py-Im polyamides directly caused apoptosis in tumor cells and retarded cell migration in the absence of T cells through inhibiting the Akt/caspase-3 pathway. Also, in a coculture system, they enhanced the T-cell-mediated killing of tumor cells by the reversal of immune escape. Because such polyamides induced antitumor effects via both immune and nonimmune pathways, they could be further developed as promising PD-L1 gene-targeting antitumor drugs.

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T cells modified to co-express cytokine or other factors with chimeric antigen receptor (CAR) can induce substantial and persistent increases in antitumor capacity in vivo. However, the uncontrolled expression of cytokines or factors can lead to the overactivation of immune cells, causing severe adverse events such as cytokine release syndrome (CRS) and neurotoxicity by CAR T cells with excessive growth potential. Conventional promoters are unregulated, and their expression is unlimited in T cells. In this study, by connecting the cytomegalovirus (CMV) enhancer, core interferon gamma (IFN-γ) promoter, and a T-lymphotropic virus long terminal repeat sequence (TLTR), we constructed and screened the chimeric promoter CIFT, which was highly expressed in some cell lines secreting IFN-γ and silenced in others. We placed this promoter upstream of the anti-programmed cell death protein 1 (anti-PD-1) antibody gene, and this construct was co-transfected with the CAR construct into T cells. In vitro or in vivo, CAR T cells showed increased secretion of anti-PD-1 antibody under control of the chimeric promoter CIFT. pS-CIFT-αPD-1/CAR T also had similar or lower PD-1 expression, higher levels of T cell activation, more release of IFN-γ, and better antitumor activity specifically against mesothelin-positive and PD-1 ligand 1 (PD-L1)-positive cell lines. The chimeric promoter may be a promising strategy to manipulate the content of immune checkpoint inhibitors or other proteins in future therapeutic approaches for cancer treatment.

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G-quadruplexes are non-canonical four stranded secondary structures possessing great biological importance. Controlling G-quadruplex conformation for further regulating biological processes is both exciting and challenging. In this study, we described a method for regulating G-quadruplex conformation by click chemistry for the first time. 8-ethynyl-2'-deoxyguanosine was synthesized and incorporated into a 12-nt telomere DNA sequence. Such a sequence, at first, formed mixed parallel/anti-parallel G-quadruplexes, while it changed to anti-parallel after reaction with azidobenzene. Meanwhile, the click reaction can give the sequence intense fluorescence.

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Cooperation between pairs of transcription factors (TFs) has been widely demonstrated to play a pivotal role in the spatiotemporal regulation of gene expression, but blocking cooperative TF pair-DNA interactions synergistically has been challenging. To achieve this, we designed programmable DNA binder pyrrole-imidazole polyamides conjugated to host-guest assemblies (Pip-HoGu) to mimic the cooperation between natural TF pairs. By incorporating cyclodextrin (Cyd)-adamantane (Ada), we synthesized Ada1 (PIP1-Ada) and Cyd1 (PIP2-Cyd), which were evaluated using Tm, EMSA, competitive, and SPR assays and molecular dynamics studies. The results consistently demonstrated that Pip-HoGu system formed stable noncovalent cooperative complexes, thereby meeting key criteria for mimicking a TF pair. The system also had a longer recognition sequence (two-PIP binding length plus gap distance), favorable sequence selectivity, higher binding affinity, and in particular, a flexible gap distance (0-5 bp). For example, Ada1-Cyd1 showed thermal stability of 7.2 °C and a minimum free energy of interaction of -2.32 kcal·mol-1 with a targeting length of 14 bp. Furthermore, cell-based evaluation validated the capability of Pip-HoGu to exhibit potent cooperative inhibitory effects on gene expression under physiological conditions by disrupting TF pair-DNA function. In conclusion, the modular design of Pip-HoGu defines a general framework for mimicking naturally occurring cooperative TF pair-DNA interactions that offers a promising strategy for applications in the precise manipulation of cell fate.

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Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit.

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With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a ß-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py-Im polyamides or between Py-Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a KD value of 16nM was recorded. Polyamide 5, containing a ß-alanine-ß-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py-Im polyamide compounds.

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N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are organic molecules that can recognize predetermined DNA sequences in a sequence-specific manner. Human telomeres contain regions of (TTAGGG)n repetitive nucleotide sequences at each end of chromosomes, and these regions protect the chromosome from deterioration or from fusion with neighboring chromosomes. The telomeres are disposable buffers at the ends of chromosomes that are truncated during cell division. Tandem hairpin Py-Im polyamide TH59, which recognizes human telomere sequences, was reported by Laemmli's group in 2001. Here, we synthesized three types of Py-Im polyamides 1-3 based on TH59 for specific recognition of human telomere repeat sequences. Thermal melting temperature (Tm) measurements and surface plasmon resonance analysis were used to evaluate the abilities of the three types of Py-Im polyamides to discriminate between three kinds of DNA sequences. Significantly, the results showed that polyamides 1 and 2 have better affinities to TTAAGG than to TTAGGG. In contrast, polyamide 3 displayed good specificity to human telomere sequence, TTAGGG, as expected on the basis of Py-Im binding rules.

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In order to develop an optimal subunit as a T-recognition element in hairpin polyamides, 15 novel chirality-modified polyamides containing (R)-α,ß-diaminopropionic acid ((R) ß α-NH 2), (S)-α,ß-diaminopropionic acid ((S) ß α-NH 2), (1R,3S)-3-aminocyclopentanecarboxylic acid ((RS) Cp), (1S,3R)-3-amino-cyclopentanecarboxylic acid ((RS) Cp), (1R,3R)-3-aminocyclopentanecarboxylic acid ((RR) Cp) and (1S,3S)-3-amino-cyclopentanecarboxylic acid ((SS) Cp) residues were synthesized. Their binding characteristics to DNA sequences 5'-TGCNCAT-3'/3'-ACGN'GTA-5' (N⋅N'=A⋅T, T⋅A, G⋅C and C⋅G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4, AcIm-(S) ß α-NH 2-ImPy-γ-ImPy-ß-Py-ßDp (ß/(S) ß α-NH 2 pair), bound to a DNA sequence containing a core binding site of 5'-TGCACAT-3' with a dissociation equilibrium constant (K(D) ) of 4.5×10(-8) m. This was a tenfold improvement in specificity over 5'-TGCTCAT-3' (K(D) =4.5×10(-7) M). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3-aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 ((RR) Cp/ß pair) had a 15-fold binding preference for 5'-TGCTCAT-3' over 5'-TGCACAT-3'. A large difference in standard free energy change for A⋅T over T⋅A was determined (ΔΔG(o) =5.9 kJ mol(-1) ), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry (9 to 5'-TGCTCAT-3'/3'-ACGAGTA-5') was shown by MSim to be optimal for the chiral five-membered cycle to fit the minor groove. Collectively, the study suggests that the (S)-α-amino-ß-aminopropionic acid and (1R,3R)-3-aminocyclopentanecarboxylic acid can serve as a T-recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)-3-aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides.

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