The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) can alter gene-expression. However, approaches that allow to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells, revealed that G4s fluctuate between folded and unfolded states. We also demonstrated that G4-formation in live cells is cell-cycle dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4-formation in living cells.

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

The discovery of protein ligands,capable of forming ar eversible covalent bond with amino acid residues on ap rotein target of interest, may represent ag eneral strategy for the discovery of potent small-molecule inhibitors.W e analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 HNMR techniques.2 -Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range.T hese benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in as ingle small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with amolar excess of hydroxylamine. Conflict of interestD.N. is ac o-founder and shareholder of Philogen (www.philogen.com), aSwiss-Italian Biotech company that operates in the field of DNA-Encoded Chemical Libraries.J .S.isaboard member of Philochem AG (www.philochem.ch).