Compelling evidence shows that amino acids can influence tumor-immune cell communication. Their transfer across the plasma membrane involves multiple amino acid transporters within the solute carrier (SLC) family. Here, we present a new strategy to modulate SLC function by targeting the SLC G-quadruplexes (G4s). We first screen eight SLC genes in the promoter region and identify G4s, including one in the promoter region of glutamine transporter SLC38A2. Then, we design a CRISPR-chiral metallohelix system that selectively targets and stabilizes the SLC38A2 G4 in tumor cells, demonstrating evident enantioselectivity with the Λ-enantiomer being more effective. This leads to SLC38A2 downregulation, which shifts glutamine uptake from tumor cells to dendritic cells (DCs), thereby redirecting glutamine metabolism to activate DCs. Furthermore, we construct a CRISPR-DHX36 system to unwind the SLC38A2 G4 in DCs and upregulate SLC38A2 expression, further augmenting glutamine uptake to compete with tumor cells. The dual CRISPR system with the ability to "fold" and "unfold" the G4 motif in different cell types leads to a marked enhancement of CD8+ T cell activation and killing ability. Therefore, our work sheds new light on cancer immune regulation by modulating the metabolic communication between cancer cells and immune cells.

The emergence of numerous variants of SARS-CoV-2 still presents the major challenges in the fight against this disease by reducing the efficacy of vaccines and drugs. RNA G-quadruplexes (G4s) in the SARS-CoV-2 genome are highly conserved and have thus been spotlighted as a promising therapeutic target to combat a wider range of variants. However, very few RNA G4 specific compounds have been reported. Here, a small library of 64 chiral metallohelices has been constructed for screening SARS-CoV-2 G4-specific binders. After screening, we found that one pair of the enantiomers showed the best enantioselectivity. The Λ enantiomer can strongly stabilize SARS-CoV-2 G4s, inhibit the expression of virus protein, and reduce the SARS-CoV-2 RNA copies and viral titers in Vero E6 cells. In contrast, the Δ enantiomer has much weaker effects than the Λ enantiomer under the same experimental conditions, showing an obvious enantioselectivity. Further studies indicate that the Λ enantiomer prefers binding to SARS-CoV-2 G4s rather than binding to the single/double-stranded DNA and commonly reported human DNA G4s, indicating its selectivity to RNA G4s. This work provides the first example for enantioselectively targeting SARS-CoV-2 G4s, and will promote developing drug candidates for targeting virus G4s.

The Mpox virus (MPXV) has emerged as a formidable orthopoxvirus, posing an immense challenge to global public health. An understanding of the regulatory mechanisms of MPXV infection, replication and immune evasion will benefit the development of novel antiviral strategies. Despite the involvement of G-quadruplexes (G4s) in modulating the infection and replication processes of multiple viruses, their roles in the MPXV life cycle remain largely unknown. Here, we found a highly conservative and stable G4 in MPXV that acts as a positive regulatory element for viral immunodominant protein expression. Furthermore, by screening 42 optically pure chiral metal complexes, we identified the Λ enantiomer of a pair of chiral helical compounds that can selectively target mRNA G4 and enhance expression of the 39-kDa core protein encoded by the MPXV A5L gene. Mechanistically, RNA G4-specific helicase DHX36 inhibits A5L protein expression by unwinding G4s. In contrast, MH3 Λ enhanced mRNA stability by specifically targeting G4 structures and subsequently increased protein expression. Furthermore, given the pivotal role of the 39-kDa core protein in activating immune responses and facilitating virion maturation, modulation of MPXV G4 folding by MH3 Λ exhibited inhibitory effects on MPXV replication through enhancing the immune response. Our findings underscore the critical involvement of G4 in the MPXV life cycle and offer potential avenues for developing antiviral drugs that target G4.

As an important pluripotency transcription factor, Sox2 plays key roles in the stemness maintenance of cancer stem‐like cells (CSCs). Here, we found that a highly conserved G‐rich region surrounding transcriptional start sites (TSSs) of Sox2 gene can form G‐quadruplex (Sox2G4) both in vitro and in cells. More intriguingly, the glycoconjugated triplex metallohelice [Fe 2 L Δ‐f 3 ] 4+ (Δ‐f) can enantioselectively stabilize Sox2G4. Sox2G4 stabilization induced by [Fe 2 L Δ‐f 3 ] 4+ (Δ‐f) rather than Λ‐f results in inhibition of Sox2 expression both in mRNA and protein levels in breast CSCs and decreases the cell viability and stemness of breast CSCs. These results indicate that [Fe 2 L Δ‐f 3 ] 4+ (Δ‐f) has the enantioselectivity even in living cells. In vivo studies further demonstrate that Δ‐f can reduce tumor initiation of breast CSCs. To our knowledge, this is the first report that targeting G‐quadruplexes of pluripotency genes can regulate the fates of CSCs. Our work provides a new way to target CSC gene and eradicate cancer stem cells.

Highly diastereoselective self-assembly reactions give both enantiomers (Λ and Δ) of anti-parallel triple-stranded bimetallic Co(ii) and Co(iii) cationic helices, without the need for resolution; the first such reaction for Co. The complexes are water soluble and stable, even in the case of Co(ii). Studies in a range of cancer and healthy cell lines indicate high activity and selectivity, and substantial differences between enantiomers. The oxidation state has little effect, and correspondingly, Co(iii) compounds are reduced to Co(ii) e.g. by glutathione. In HCT116 colon cancer cells the Λ enantiomer induces dose-dependent G2-M arrest in the cell cycle and disrupts microtubule architectures. This Co(ii) Λ enantiomer is ca. five times more potent than the isostructural Fe(ii) compound. Since the measured cellular uptakes are similar this implies a higher affinity of the Co system for the intracellular target(s); while the two systems are isostructural they have substantially different charge distributions as shown by calculated hydrophobicity maps. In contrast to the Λ enantiomer, Δ-Co(ii) induces G1 arrest in HCT116 cells, efficiently inhibits the topoisomerase I-catalyzed relaxation of supercoiled plasmid DNA, and, unlike the isostructural Fe(ii) system, causes DNA damage. It thus seems very likely that redox chemistry plays a role in the latter.

Targeted protein degradation has demonstrated the power to modulate protein homeostasis. For overcoming the limitation to intracellular protein degradation, lysosome targeting chimeras have been recently developed and successfully utilized to degrade a range of disease-relevant extracellular and membrane proteins. Inspired by this strategy, here we describe our proof-of-concept studies using metallohelix-based degraders to deliver the extracellular human islet amyloid polypeptide (hIAPP) into the lysosomes for degradation. Our designed metallohelix can bind and inhibit hIAPP aggregation, and the conjugated tri-GalNAc motif can target macrophage galactose-type lectin 1 (MGL1), yielding chimeric molecules that can both inhibit hIAPP aggregation and direct the bound hIAPP for lysosomal degradation in macrophages. Further studies demonstrate that the enhanced hIAPP clearance has been through the endolysosomal system and depends on MGL1-mediated endocytosis. Intriguingly, Λ enantiomers show even better efficiency in preventing hIAPP aggregation and promoting internalization and degradation of hIAPP than Δ enantiomers. Moreover, metallohelix-based degraders also faciltate the clearance of hIAPP through asialoglycoprotein receptor in liver cells. Overall, our studies demonstrate that chiral metallohelix can be employed for targeted degradation of extracellular misfolded proteins and possess enantioselectivity.

A novel photoactivatable Pt(IV) diazido anticancer agent, Pt-succ-DFO, bearing a pendant deferoxamine (DFO) siderophore for radiometal chelation, has been synthesized for the study of its in vivo behavior with radionuclide imaging. Pt-succ-DFO complexation of Fe(III) and Ga(III) ions yielded new heterobimetallic complexes that maintain the photoactivation properties and photocytotoxicity of the parent Pt complex in human cancer cell lines. Radiolabeled Pt-succ-DFO-68Ga (t1/2 = 68 min, positron emitter) was readily prepared under mild conditions and was stable in the dark upon incubation with human serum. PET imaging of Pt-succ-DFO-68Ga in healthy mice revealed a promising biodistribution profile with rapid renal excretion and limited organ accumulation, implying that little off-target uptake is expected for this class of agents. Overall, this research provides the first in vivo imaging study of the whole-body distribution of a photoactivatable Pt(IV) azido anticancer complex and illustrates the potential of radionuclide imaging as a tool for the preclinical development of novel light-activated agents.

Self-assembled enantiomers of an asymmetric di-iron metallohelix differ in their antiproliferative activities against HCT116 colon cancer cells such that the compound with Λ-helicity at the metals becomes more potent than the Δ compound with increasing exposure time. From concentration- and temperature-dependent 57Fe isotopic labelling studies of cellular accumulation we postulate that while the more potent Λ enantiomer undergoes carrier-mediated efflux, for Δ the process is principally equilibrative. Cell fractionation studies demonstrate that both enantiomers localise in a similar fashion; compound is observed mostly within the cytoskeleton and/or genomic DNA, with significant amounts also found in the nucleus and membrane, but with negligible concentration in the cytosol. Cell cycle analyses using flow cytometry reveal that the Δ enantiomer induces mild arrest in the G1 phase, while Λ causes a very large dose-dependent increase in the G2/M population at a concentration significantly below the relevant IC50. Correspondingly, G2-M checkpoint failure as a result of Λ-metallohelix binding to DNA is shown to be feasible by linear dichroism studies, which indicate, in contrast to the Δ compound, a quite specific mode of binding, probably in the major groove. Further, spindle assembly checkpoint (SAC) failure, which could also be responsible for the observed G2/M arrest, is established as a feasible mechanism for the Λ helix via drug combination (synergy) studies and the discovery of tubulin and actin inhibition. Here, while the Λ compound stabilizes F-actin and induces a distinct change in tubulin architecture of HCT116 cells, Δ promotes depolymerization and more subtle changes in microtubule and actin networks.

Naturally occurring peptides in many living systems perform antimicrobial and anticancer host defence roles, but their potential for clinical application is limited by low metabolic stability and relatively high costs of goods. Self-assembled helical metal complexes provide an attractive synthetic platform for non-peptidic architectures that can emulate some of the properties of short cationic α-helical peptides, with tuneable charge, shape, size and amphipathicity. Correspondingly there is a growing body of evidence demonstrating that these supramolecular architectures exhibit bioactivity that emulates that of the natural systems. We review that evidence in the context of synthetic advances in the area, driven by the potential for biomedical applications. We note some design considerations for new biologically-relevant metallohelices, and give our outlook on the future of these compounds as therapeutic peptidomimetics.

Functionalised triazole aldehydes are used in the highly selective self-assembly of water-compatible, optically pure, low symmetry Fe(ii)- and Zn(ii)-based metallohelices. Sub-micromolar antiproliferative activity is observed against various cancerous cell lines, accompanied by excellent selectivityversusnon-cancerous cells and potential for synergistic combinatorial therapy with cisplatin.