Progress in understanding crystallisation: a personal perspective

After this Discussion meeting, most participants felt that we do not understand crystallisation. However, in the 1980s, I believe that most scientists would have considered that crystallisation was adequately understood. These concluding remarks give a personal impression of the progress that has been made towards appreciating the complexity of crystallisation over the past forty years

A predicted dimer-based polymorph of 10,11-dihydrocarbamazepine (Form IV)

A novel polymorph of 10,11-dihydrocarbamazepine (form IV), which had been predicted to be thermodynamically feasible, was obtained from the vapour phase and displays an R22(8) hydrogen bonded dimer motif in contrast to the catemeric motifs in forms I–III

Isomorphous template induced crystallisation : a robust method for the targeted crystallisation of computationally predicted metastable polymorphs

A new method of inducing the crystallisation of metastable polymorphs by isomorphous templating has been developed and used to reproduce the crystallisation of CBZ-V on the surface of DHC-II. Studies of the growth of CBZ-V on DHC-II single crystals show crystals growing laterally and vertically on DHC-II surfaces without any significant face selectivity. The generality of this computationally inspired crystallisation approach is demonstrated by producing the first crystals of an entirely new polymorph of cyheptamide, which is isomorphous to both DHC-II and CBZ-V

Palonosetron-5-HT3Receptor Interactions As Shown by a Binding Protein Cocrystal Structure.

Palonosetron is a potent 5-HT3receptor antagonist and an effective therapeutic agent against emesis. Here we identify the molecular determinants of compound recognition in the receptor binding site by obtaining a high resolution structure of palonosetron bound to an engineered acetylcholine binding protein that mimics the 5-HT3receptor binding site, termed 5-HTBP, and by examining the potency of palonosetron in a range of 5-HT3receptors with mutated binding site residues. The structural data indicate that palonosetron forms a tight and effective wedge in the binding pocket, made possible by its rigid tricyclic ring structure and its interactions with binding site residues; it adopts a binding pose that is distinct from the related antiemetics granisetron and tropisetron. The functional data show many residues previously shown to interact with agonists and antagonists in the binding site are important for palonosetron binding, and indicate those of particular importance are W183 (a cation-π interaction and a hydrogen bond) and Y153 (a hydrogen bond). This information, and the availability of the structure of palonosetron bound to 5-HTBP, should aid the development of novel and more efficacious drugs that act via 5-HT3receptors.Supported by a grant from the MRC (MR/L02/676) to S.C.R.L

Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP.

Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.Supported by grants from the Wellcome Trust (81925) and the MRC to S.C.R.L.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/cn500369

On the Application of Strong Magnetic Fields during Organic Crystal Growth

We investigate the effect of crystal growth within a magnetic field for three polymorphic pharmaceuticals, using an experiment where the magnetic field can be varied in strength without altering other crystallization conditions. In the case of carbamazepine, fields above 0.6 T produce metastable form I, and for flufenamic acid, there is an increased propensity to crystallize metastable form I around 1 T. In contrast, the magnetic field has no effect on the crystallization of mefenamic acid, a closely related molecule. The growth of the metastable β polymorph of coronene within a magnetic field at ambient temperature is difficult to reproduce but has been seen as a minor component, consistent with this transformation to the more stable form being facile, depending on the particle size. Calculations of the diamagnetic susceptibility tensors of the polymorphs and their morphologies provide semiquantitative estimates of how the diamagnetic susceptibilities of crystallites differ between polymorphs and explain why mefenamic acid crystallization is unaffected. As the onset of crystallization of carbamazepine and coronene, as defined by changes in turbidity, occur at lower temperatures and hence greater supersaturations in certain ranges of magnetic field strength, this suggests that the field causes precipitation of the metastable form through Ostwald’s rule of stages

Predictors of dominance rank and agonistic interactions in captive Livingstone’s fruit bats

Male dominance hierarchies have been studied in many animals but rarely in bats (Chiroptera). The dominance rank of social animals may dictate access to resources and mates; therefore, it has important implications for an individual’s fitness and is crucial for successful captive management. Between January and December 2018, at both Bristol Zoo Gardens (Bristol, UK) and Jersey Zoo (Jersey, British Isles), we observed 19 male Livingstone’s fruit bats Pteropus livingstonii using focal follows for 345 h overall, noting the outcome of all agonistic interactions. We recorded instigators of interactions, along with winners and losers, and analyzed these data using the R-package “EloRating” to create Elo-rating temporal plots of dominance ranks. We used generalized linear mixed models and multiple linear regression to analyze interaction data and test hypotheses regarding predictors of dominance rank, frequency of agonistic interaction, and choice of interaction partner. Age was positively correlated with dominance rank up to around year 9, when an asymptote was attained. Highly ranked bats instigated the most agonistic interactions, and largely directed these interactions at bats with much lower rankings than themselves. Hierarchies were extremely stable throughout the data collection period at both sites. We conclude that Livingstone’s fruit bats have a stable linear dominance hierarchy, with high-ranking, typically older males instigating the most interactions with lowest ranking males to secure dominance rank. This study adds to the limited discourse on Pteropus social behaviors, indicating that some bat species may have social systems similar in complexity to some nonhuman primates.<br/

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation.

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6'M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6'M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6' residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.This project was supported by the Wellcome Trust (grant 81925 to SCRL). SCRL is a Wellcome Trust Senior Research Fellow in Basic Biomedical Studies.This is the final published version, which first appeared at http://www.sciencedirect.com/science/article/pii/S0965174814001684

Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine₃ Receptor Revealed by Unnatural Amino Acid Mutagenesis

The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HT₃ receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HT₃ receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors