Recently, a novel negative allosteric modulator (NAM) of the D-like dopamine receptors 1 was identified through virtual ligand screening. This ligand comprises a thieno[2,3- d]pyrimidine scaffold that does not feature in known dopaminergic ligands. Herein, we provide pharmacological validation of an allosteric mode of action for 1, revealing that it is a NAM of dopamine efficacy and identify the structural determinants of this allostery. We find that key structural moieties are important for functional affinity and negative cooperativity, while functionalization of the thienopyrimidine at the 5- and 6-positions results in analogues with divergent cooperativity profiles. Successive compound iterations have yielded analogues exhibiting a 10-fold improvement in functional affinity, as well as enhanced negative cooperativity with dopamine affinity and efficacy. Furthermore, our study reveals a fragment-like core that maintains low μM affinity and robust negative cooperativity with markedly improved ligand efficiency.

The D dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D receptor agonists possess known clinical liabilities. We discovered two structurally distinct D receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and -arrestin-mediated signaling and increase the affinity of dopamine for the D receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both -arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D receptor as well as the development of optimized lead compounds for therapeutic use.

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side-effects (EPS) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D 2 receptor (D 2 R) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the D 2 R whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the D 2 R predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side-effects independent of its D 2 R action. Our results suggest an optimal kinetic profile for D 2 R antagonist APDs that avoids EPS. To begin exploring this hypothesis, we conducted a structure-kinetic relationship study of haloperidol and reveal that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimisation of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles.  INTRODUCTIONHaloperidol (1, Figure 1) is an effective, typical antipsychotic drug (APD) used in the treatment of schizophrenia (SCZ). As for all current APDs, its mechanism of action is primarily through antagonism of dopamine (DA) D 2 receptors (D 2 R) in the mesolimbic pathway, where excessive DA activity is thought to underlie the positive symptoms of schizophrenia. [1][2][3] Unfortunately, 1 along with

The adenosine A 1 receptor is a therapeutic target based on its ability to provide cardioprotection during episodes of myocardial ischemia and reperfusion injury. However, the clinical translation of A 1 R agonists has been hindered by dose-limiting adverse effects (bradycardia and hypotension). Previously, we demonstrated that the bitopic agonist VCP746 (1), consisting of an adenosine pharmacophore linked to an allosteric moiety, can stimulate cardioprotective A 1 R signaling effects in the absence of unwanted bradycardia. This study maps the structure−activity relationships of 1 through modifications to the linker moiety. Derivatives differing in the flexibility, length, and nature of the linker were assessed, which revealed that the linker is tolerant of several modifications including added rigidity. Ligands featuring 1,4-disubstituted 1,2,3-triazoles were the most biased of the novel analogues but also displayed sub-nanomolar potency in a cAMP accumulation assay at the A 2B R. To our knowledge, 10 is the most potent A 2B R agonist published to date.

(1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively); (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with superior allosteric properties.

We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D 2 receptor (D 2 R) based on thieno [2,3-d]pyrimidine 1, showing it can be structurally simplified to reveal low molecular weight, fragment-like NAMs that retain robust negative cooperativity, such as 3. Herein, we report the synthesis and functional profiling of analogues of 3, placing specific emphasis on examining secondary and tertiary amino substituents at the 4-position, combined with a range of substituents at the 5/6-positions (e.g. aromatic/aliphatic carbocycles). We identify analogues with diverse pharmacology at the D 2 R including NAMs (19fc) with sub-µM affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i).