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There were an estimated 249 million cases of malaria globally in 2022, causing approximately 608 000 deaths. Most of these are attributed to infection by P. falciparum . Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms, including P. falciparum . A panel of 25 S-MGBs was synthesised, including those bearing an amidine tail group, and their antiplasmodial activity against 3D7 and Dd2 strains was determined in vitro using an asexual P. falciparum imaging assay. Determination of activity against HEK293 cells allowed for selective cytotoxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. A comparison of 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) potency showed no evidence of cross-resistance across the S-MGB set. S-MGB-356 , S-MGB-368 and S-MGB-359 , amidine tail containing S-MGBs, were identified as the most promising hit compounds based on their selectivity indices (HEK293/3D7) of >612.6, >335.8 and >264.8, respectively. S-MGB-356 , S-MGB-368 and S-MGB-359 were confirmed to bind to DNA as dimers, with gDNA thermal shifts (Δ T m ) of 12 °C, 3 °C and 16 °C, respectively. Together, these data demonstrate that amidine tail bearing S-MGBs are promising hit compounds against P. falciparum , and can be further optimised into lead compounds., Competing Interests: The authors declare the following competing financial interest(s): MP, LMCM, AIK, CJS and FJS are part of revenue sharing agreements with their University relating to the Strathclyde minor groove binder project. Additionally, CJS and FJS have financial interests through shares in the company, Rostra Therapeutics. All other authors: none to declare., (This journal is © The Royal Society of Chemistry.)

There is mounting evidence that crystal nucleation from supersaturated solution involves the formation and reorganization of prenucleation clusters, contradicting classical nucleation theory. One of the key unresolved issues pertains to the origin, composition, and structure of these clusters. Here, a range of amino acids and peptides is investigated using light scattering, mass spectrometry, and in situ terahertz Raman spectroscopy, showing that the presence of amorphous aggregates is a general phenomenon in supersaturated solutions. Significantly, these aggregates are found on a vast range of length scales from dimers to 30-mers to the nanometre and even micrometre scale, implying a continuous distribution throughout this range. Larger amorphous aggregates are sites of spontaneous crystal nucleation and act as intermediates for laser-induced crystal nucleation. These results are shown to be consistent with a nonclassical nucleation model in which barrierless (homogeneous) nucleation of amorphous aggregates is followed by the nucleation of crystals from solute-enriched aggregates. This provides a novel perspective on crystal nucleation and the role of nonclassical pathways., Competing Interests: The authors declare no conflicts of interests., (This journal is © The Royal Society of Chemistry.)

In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN : DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15 : DDA1 : DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15 : DDA1 : DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1 : 1 : 1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and its self-association into dimers and trimers at reduced salt concentration during structural analysis.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs ( N ε -methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for semi-quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the p K a of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution-phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy, the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for semi-quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process., Competing Interests: F. J. S. and C. J. S. are inventors on patents that pertain to S-MGBs, filed by the University of Strathclyde, and may benefit financially from commercial efforts relating to these patents., (This journal is © The Royal Society of Chemistry.)

In the last ten years mass spectrometry has emerged as a powerful biophysical technique capable of providing unique insights into the structure and dynamics of proteins. Part of this explosion in use involves investigations of the most recently 'discovered' subset of proteins: the so-called 'Intrinsically Disordered' or 'Natively Unstructured' proteins. A key advantage of the use of mass spectrometry to study intrinsically disordered proteins (IDPs) is its ability to test biophysical assertions made about why they differ from structured proteins. For example, from the charge state distribution presented by a protein following nano-electrospray (n-ESI) it is possible to infer the range of conformations present in solution and hence the extent of disorder; n-ESI is highly sensitive to the degree of folding at the moment of transfer from the liquid to the gas phase. The combination of mass spectrometry with ion mobility (IM-MS) provides rotationally averaged collision cross-sections of molecular ions which can be correlated with conformation; this too can be applied to IDPs. Another feature which can be monitored by IM-MS is the tendency of disordered proteins to form amyloid fibrils, the protein aggregates involved in the onset of neurodegenerative diseases such as Parkinson's and Alzheimer's. IM-MS provides a useful insight into events that occur during the early stages of aggregation including delineating the structure of the monomer, identifying oligomer distributions, and revealing mechanistic details of the aggregation process. Here we will review the use of MS and IM-MS to study IDPs using examples from our own and other laboratories.