The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR’s constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.

The discovery of targeted covalent inhibitors is of increasing importance in drug discovery. Finding efficient covalent binders requires modulation of warhead reactivity and optimisation of warhead geometry and non-covalent interactions. Uncoupling the contributions that these factors make to potency is difficult and best practice for a testing cascade that is pragmatic and informative is yet to be fully established. We studied the structure-reactivity-activity relationships of a series of analogues of the EGFR inhibitor poziotinib with point changes in two substructural regions as well as variations in warhead reactivity and geometry. This showed that a simple probe displacement assay that is appropriately tuned in respect of timing and reagent concentrations can reveal structural effects on all three factors: non-covalent affinity, warhead reactivity and geometry. These effects include the detection of potency differences between an enantiomeric pair that differ greatly in their activity and their capacity to form a covalent bond. This difference is rationalised by X-ray crystallography and computational studies and the effect translates quantitatively into cellular mechanistic and phenotypic effects.

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA₁ induces auto-phosphorylation of MenT₁ by repositioning the MenT₁ phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA₃ is similarly able to induce auto-phosphorylation of cognate toxin MenT₃. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.

Per–Arnt–Sim (PAS) domain kinase (PASK) is a conserved metabolic sensor that modulates the activation of critical proteins involved in liver metabolism and fitness. However, despite its key role in mastering the metabolic regulation, the molecular mechanism of PASK’s activity is ongoing research, and structural information of this important protein is scarce. To investigate this, we integrated structural bioinformatics with state-of-the-art modeling and molecular simulation techniques. Our goals were to address (1) how many regulatory PAS domains PASK is likely to have, (2) how those domains modulate the kinase activity, and (3) how those interactions could be controlled by small molecules. Our results indicated the existence of three N-terminal PAS domains. Solvent mapping and fragment docking identified a consensus set of ‘druggable hot spots’ within all domains, as well as at domain–domain interfaces. Those ‘hot spots’ could be modulated with chemically diverse small molecular probes, which may serve as a starting point for rationally designed therapeutics modulating these specific sites. Our results identified a plausible mechanism of autoinhibition of kinase activity, suggesting that all three putative PAS domains may be required. Future work will focus on validation of the predicted PASK models and development of small-molecule inhibitors of PASK by targeting its ‘druggable hot spots’.

Type I collagen is the main structural protein of vertebrates and forms molecular trimers from the COL1A1 and COL1A2 gene products: proα1α(I) and proα2α(I), during biosynthesis. Calcium ions are required for trimers to form. The amino acid sequence of the C-propeptide of collagen, which is removed before collagen fibril formation, initially drives heterotrimerisation. Abnormal homotrimeric type I collagen is associated with age-related diseases including cancer, fibrosis, musculoskeletal and cardiovascular conditions but the circumstances under which the homotrimer may form are poorly understood. Here we used molecular dynamics simulations of the C-propeptide protein structure to show that inter- and intra-chain hydrogen bonding is affected by loss of calcium and that this leads chains to become destabilised, particularly at the interfaces of each chain. Loss of calcium resulted increased distances between the cysteine residues that form inter-chain disulphide bonds, preventing the formation of these bonds. Pulling simulations and modelling of calcium dissociation from monomers showed that calcium ions were more strongly bound to the α1(I) than the α2(I) chain. However, enhanced sampling methods implied the α2(I) chain has a higher trimer affinity than a third α1(I) chain in the presence of structural calcium. To quantify assembly thermodynamics, we computed relative binding free energies by alchemical thermodynamic integration, demonstrating that α2(I)-specific residues at the interchain interface conferred a measurable thermodynamic advantage to trimer formation in the presence of calcium. Hence although heterotrimerisation is normally favoured, in reduced calcium conditions the homotrimer can form by sequestering available calcium to the α1(I) chains. This study provides a molecular explanation for a calcium-based mechanism driving heterotrimerisation versus homotrimerisation of type I collagen.

The electron shuttle and coenzyme nicotinamide adenine nucleotide (NAD) is essential for cellular metabolism and homeostasis. NAD levels significantly fluctuate in cells, whilst ageing and age-related diseases are associated with depletion of this metabolite. However, how NAD changes are monitored by nutrient/energy sensing signalling pathways remains poorly understood. We found that at physiological concentrations NAD controls the activity of the AMP-activated protein kinase (AMPK) in vitro and in human cells. Mechanistically, NAD binds gamma subunit of AMPK, and mutagenesis of the putative binding site renders the holoenzyme insensitive to NAD inhibition. Hyperactivation of AMPK in response to NAD depletion suppresses metabolic pathways including mammalian Target of Rapamycin Complex I (mTORC1) and autophagy. These results demonstrate that in addition to monitoring cellular energy levels AMPK functions as a NAD sensor, providing novel insight into how cells and tissues detect and respond to metabolic fluctuations with implications for stress resistance and ageing.