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Belumosudil is a selective Rho‐associated coiled‐coil containing protein kinase 2 (ROCK2) inhibitor. ROCK2 has been shown to drive proinflammatory response and fibrosis that occurs with chronic graft‐versus‐host disease; therefore, inhibition of ROCK2 has emerged as a therapeutic target for chronic graft‐versus‐host disease. In this phase 1 two‐part study, the pharmacokinetics, mass balance, and metabolic profile of belumosudil were evaluated after single doses of unlabeled belumosudil oral tablets (200 mg), radiolabeled belumosudil intravenous (IV) microtracer infusions (100 μg), and radiolabeled oral capsules (200 mg). Absolute bioavailability based on area under the plasma concentration–time curve from time 0 to infinity for the oral dose/area under the plasma concentration–time curve from time 0 to infinity for the IV dose was calculated as 63.7%. Radiolabeled IV microtracer dosing demonstrated a low extraction ratio and distribution of belumosudil into tissues. The majority of total radioactivity was recovered in feces, with minimal amounts recovered in urine, suggesting minimal renal elimination of belumosudil. In addition to parent and main metabolite KD025m2, metabolites identified in plasma included the phase 2 metabolites O‐dealkylated belumosudil sulfate and belumosudil glucuronide. These metabolites (with the exception of the glucuronide) in addition to monohydroxy‐belumosudil, and belumosudil diol were identified in feces. No metabolites in urine accounted for >10% of the radioactive dose. [ABSTRACT FROM AUTHOR]

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition to determine functionality. The results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.

A HEK-293 cell line expressing mouse 5-H[T.sub.3A]Rs containing a C-terminal extension that confers high-affinity binding of [alpha]-bungarotoxin ([alpha]BgTx) is established ([alpha]BgTx-5-H[T.sub.3A]Rs) and is used for purifying [alpha]BgTx-5-H[T.sub.3A]Rs in a lipid environment for use in structural studies by using photoaffinity labeling. The results have described the surface of the 5-H[T.sub.3A]RM4 helix that is exposed to lipid and has established that the surface is equivalent to the surface exposed to lipid in the Torpedo nicotinic acetylcholine receptor.

The structural studies of the lipid-protein interface of purified and lipid-reconstituted human [alpha]4[beta]2 neuronal nicotinic acetylcholine receptors (nAChRs) is reported. A high degree of structural homology is obtained by using stably transfected HEK-293 cell line.

A HEK-293 cell line that stably expresses mouse 5-HT3ARs containing a C-terminal extension that confers high-affinity binding of ct-bungarotoxin (αBgTx) was established (αBgTx-5-HT3ARs) and used to purify αBgTx-5-HT3ARs in a lipid environment for use in structural studies using photoaffinity labeling. αBgTx-5-HT3ARs were expressed robustly (60 pmol of [³H]BRL-43694 binding sites (∼3 μg of receptor) per milligram of protein) and dipIayed the same functional properties as wild-type receptors (serotonin EC50 = 5.3 ± 0.04 μM). While [125I]αBgTx bound to the αBgTx-5-HT3ARs with high affinity (Kd = 11 nM), application of nonradioactive αBgTx (up to 300 μM) had no effect on serotonin-induced current responses. αBgTx-5-HT3ARs were purified on an αBgTx-derivatized affinity column from detergent extracts in milligram quantities and at ∼25% purity. The hydrophobic photolabel 3-trifluoromethyl- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted αBgTx-5-HT3ARs. [125I]TID photoincorporation into the αBgTx-5-HT3AR subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and ∼60% of incorporated 125I and 17 kDa, containing the M1 - M3 transmembrane segments. Within the M4 segment, [125I]TID labeled Ser451, equivalent to the (125I]TID-Iabeled residue Thr422 at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) α1M4 α-helix. These results provide a first definition of the surface of the 5-HT3AR M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR. [ABSTRACT FROM AUTHOR]

Abstract: Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with ∼25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the α subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a ∼20 kDa Staphylococcus aureus V8 protease fragment (αV8-20; Ser173-Glu338). To further map the labeling site, the αV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (αMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel. [Copyright &y& Elsevier]

Abstract: A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding. [Copyright &y& Elsevier]

Abstract: The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109–P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation. [Copyright &y& Elsevier]

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both a subunits and of γLeu109 and 7Tyr117 in Segment E of the complementary (-) face, with no labeling detected in the 5 subunit. For affinitypurified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR. [ABSTRACT FROM AUTHOR]

Adrenocorticotropic hormone 1-24 (ACTH[1-24]) has a similar effect as endogenous ACTH(1-39) to generate cortisol by targeting the MC2R receptor on the adrenal gland. A new investigational ACTH receptor antagonist drug is being developed to treat diseases of ACTH excess (e.g., Cushing's disease) by binding to the MC2R receptor. Administration of ACTH(1-24) was used in a Phase I clinical study to assess the ability of this drug candidate to suppress the cortisol response to ACTH stimulation. A hybrid immunoaffinity-LCMS assay measuring ACTH(1-24) with a concentration range of 10 to 400 pg/ml was developed to support the study. Consistent and acceptable A&P results were achieved. The assay development and qualification will be discussed.

Linerixibat, an oral small-molecule ileal bile acid transporter inhibitor under development for cholestatic pruritus in primary biliary cholangitis, was designed for minimal absorption from the intestine (site of pharmacological action). This study characterized the pharmacokinetics, absorption, metabolism, and excretion of [ 14 C]-linerixibat in humans after an intravenous microtracer concomitant with unlabeled oral tablets and [ 14 C]-linerixibat oral solution. Linerixibat exhibited absorption-limited flip-flop kinetics: longer oral versus intravenous half-life (6-7 hours vs. 0.8 hours). The short intravenous half-life was consistent with high systemic clearance (61.9 l/h) and low volume of distribution (16.3 l). In vitro studies predicted rapid hepatic clearance via cytochrome P450 3A4 metabolism, which predicted human hepatic clearance within 1.5-fold. However, linerixibat was minimally metabolized in humans after intravenous administration: ∼80% elimination via biliary/fecal excretion (>90%-97% as unchanged parent) and ∼20% renal elimination by glomerular filtration (>97% as unchanged parent). Absolute oral bioavailability of linerixibat was exceedingly low (0.05%), primarily because of a very low fraction absorbed (0.167%; fraction escaping first-pass gut metabolism (fg) ∼100%), with high hepatic extraction ratio (77.0%) acting as a secondary barrier to systemic exposure. Oral linerixibat was almost entirely excreted (>99% recovered radioactivity) in feces as unchanged and unabsorbed linerixibat. Consistent with the low oral fraction absorbed and ∼20% renal recovery of intravenous [ 14 C]-linerixibat, urinary elimination of orally administered radioactivity was negligible (<0.04% of dose). Linerixibat unequivocally exhibited minimal gastrointestinal absorption and oral systemic exposure. Linerixibat represents a unique example of high CYP3A4 clearance in vitro but nearly complete excretion as unchanged parent drug via the biliary/fecal route. SIGNIFICANCE STATEMENT: This study conclusively established minimal absorption and systemic exposure to orally administered linerixibat in humans. The small amount of linerixibat absorbed was eliminated efficiently as unchanged parent drug via the biliary/fecal route. The hepatic clearance mechanism was mispredicted to be mediated via cytochrome P450 3A4 metabolism in vitro rather than biliary excretion of unchanged linerixibat in vivo., (Copyright © 2021 by The Author(s).)

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.