Fyfe, Tim
(2019)
Medicinal Chemistry & Chemical Biology Approaches to Investigate Prospective Treatments for CNS Diseases: Dopamine D1 Receptor Positive Allosteric Modulators, Dopamine D2 Receptor Positive & Negative Allosteric Modulators, Dopamine D2 Receptor-Ligand Kinetics.
PhD thesis, University of Nottingham.
Abstract
The dopamine (DA) D1 and D2 receptors (D1R and D2R, respectively) are G protein-coupled receptors (GPCRs) that are therapeutic targets for the symptomatic treatment of neurological disorders such as schizophrenia (SCZ) and Parkinson’s disease (PD). Classical approaches to GPCR drug discovery have focused on developing orthosteric agents with the capacity to engage the endogenous ligand (dopamine) binding site. Allosteric modulators, molecules that act via a topographically distinct but spatially linked binding site, have been touted as the next generation of CNS therapeutics and may show promise towards the treatment of SCZ and PD. To facilitate an understanding of molecular drug-receptor interactions, such small-molecule allosteric compounds may also be developed and used as biochemical tools, enabling techniques such as rational structure-based drug design or advancement of knowledge regarding key residues responsible for emerging concepts such as functional selectivity. Alternatively, the molecular basis for antipsychotic drug-induced on-target toxicities are also of significant importance in order to facilitate the development of safer therapeutics, and receptor-ligand binding kinetics is a key area of interest. Accordingly, this thesis explores multiple medicinal chemistry and chemical biology approaches to investigate: i) structural drivers of small molecule allosteric pharmacology toward understanding structure-activity-relationships (SAR) for allosteric modulator optimisation and biochemical tool development; ii) the kinetic basis for the “on target” side effect profiles of clinical antipsychotic drugs (APDs) in order to develop novel scaffolds with enhanced efficacy/side-effect profiles.
Chapter 2 comprises work published in the Journal of Medicinal Chemistry and details the synthesis of a small molecule thieno[2,3-d]pyrimidine hit arising from a virtual ligand screen (VLS) performed using the crystal structure of an antagonist bound D3R as a template. We validate the hit compound’s allosteric mode of interaction at the D2R using radioligand binding and functional assays. In addition, the synthesis and structure-activity-relationships (SARs) of a series of analogues are further explored. All compounds are evaluated via the use of an operational model of allosterism to determine values of functional affinity (KB) and negative allosteric cooperativity (α, β). Moreover, molecular docking studies were conducted using the recently determined D2R crystal structure (PDB code 6C38) to assist in elucidati ng a potential binding mode for these molecules, and provide rationale for the observed SAR. Promising analogues were identified that displayed differential attenuation of affinity/signalling efficacy, and/or increased functional binding affinity. Lastly, marked improvements in lipophilic ligand efficiency of key active analogues revealed a fragment-like starting point that can be elaborated through multiple vectors.
Chapter 3 comprises work published in the European Journal of Medicinal Chemistry and explores the synthesis and biological evaluation of an additional 36 compounds based on the core scaffold identified in the chapter 2. We assess the influence of introducing various primary and secondary amino groups, both of aliphatic and aromatic nature (e.g. pyrrolidine, aniline, cyclohexylamine) to the 4-position of the thieno[2,3-d]pyrimidine core, whilst maintaining the fused cyclohexane moiety present within the parent VLS hit (Series 1). The functional analysis of this series of compounds identified three amines (cyclobutylamino, cyclopropylamino, N,N-diethylamino) that engendered higher affinities and robust allosteric properties relative to the parent ((3-trifluoromethyl)phenyl)amino substituent. Thus, in an effort to understand their utility, these amines were further employed in conjunction various 5,6-modifications (eg., aromatic/aliphatic carbocyles). This allowed us to further explore and establish the structural determinants of D2R allosteric pharmacology and functional binding affinity. We describe the identification of analogues with a range of different functional pharmacologies, including two of the highest affinity derivatives to emerge from the investigation that maintain negative cooperativity, and surprisingly, agonists.
Chapter 4. A detailed structural understanding of allostery at the D2R will enable the progression of allosteric modulators towards the potential treatment of the symptoms of PD. This chapter investigates the SARs of a novel D2R positive allosteric modulator (PAM) as the basis for generating irreversible and fluorescent ligands to be used as biochemical tools, which may permit a better understanding of molecular allosteric interactions, and to guide rational structure-based drug design. In order to elucidate potential linking points from which we could append photoactivatable or fluorescent moieties, we designed and synthesised a focused library of analogues, initially resynthesising the parent D2R PAM using a slightly modified 11-step literature synthesis in order to further characterise its functional pharmacology, as well as making additional modifications to obtain two novel structural analogues. We also established a novel 7-step synthetic pathway to permit more efficient access to D2R PAMs, resulting in the synthesis of a further 13 structural analogues. The compounds were evaluated via the use of an operational model of allosterism to determine values of functional affinity (KB), intrinsic agonism (τB) and positive allosteric cooperativity (α, β). These parameters allowed the elucidation of the molecular determinants of allostery so that this information may be used for the design of irreversible and fluorescent biochemical tool compounds that retain their parent pharmacological profile.
Chapter 5. This chapter explores the structure-kinetic relationships of the butyrophenone APD haloperidol at the D2R for which there currently exists minimal published literature. Extrapyramidal symptoms (EPS) and hyperprolactinemia are common debilitating side-effects of typical APDs, and binding kinetics (i.e. association and dissociation rate from their biological target) at the D2R may play a role in determining these side-effect profiles. It has been suggested that a slow dissociation rate is associated with hyperprolactinemia, but that a fast association rate is associated with EPS. Our investigation further examined the ligand kinetics of butyrophenone analogues at the D2R, and how these might ultimately impact therapeutic and side-effect profiles. We assessed the ligand binding kinetics of 50 novel and literature structural analogues of the antipsychotic haloperidol using a timeresolved Förster resonance energy transfer (FRET) competition association kinetic binding assay. We determined association and dissociation rates (kon, koff), and equilibrium affinities (pKd) and discovered that the kinetic profile of the butyrophenone scaffold can vary dramatically with subtle structural modifications. This may allow optimisation of ligand kinetic parameters to design new APDs with improved side-effect profiles.
Chapter 6. The D1R has been implicated as useful target to ameliorate the cognitive deficits associated with key CNS disorders. This chapter focuses on the synthesis and pharmacological validation of D1R PAMs “compound A” and “compound B”. We conducted various chemical syntheses to generate racemic PAMs, as well as investigated the use of chiral resolving techniques and alternative chiral auxiliaries for use in asymmetric Diels-Alder chemistry toward the synthesis of optically pure enantiomers of “compound B”. The compounds were evaluated at the D1R via the use of an operational model of allosterism to yield estimates of functional affinity (KB), intrinsic agonism (τB) and allosteric positive cooperativity (α, β). All compounds displayed allosteric pharmacology. These molecules were also assessed for their subtype selectivity over the D2R (i.e. D1R vs D2R). Compound A was found to act as a competitive partial agonist, yet compound B in its racemic and enantioenriched forms, showed no activity. Compound B will now be used as a starting point to form the basis of an extensive SAR investigation into subtype selective PAMs of the D1R.
Finally, Chapter 7 provides a brief summary of the outcomes obtained from this thesis, as well as future prospects
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