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Summary: Background: Transcription factor retinoic acid‐related orphan receptor 2 (RORC2/RORγT) mediates interleukin (IL)‐17A and IL‐17F expression. IL‐17A plays a central role in the pathogenesis of several inflammatory disorders, including psoriasis. The RORC2 inhibitor PF‐06763809 has been hypothesized to inhibit IL‐17A production in T‐helper 17 (Th17) cells, thereby reducing psoriasis symptoms. Aim: To assess the safety, tolerability and effect on skin infiltrate thickness of PF‐06763809 in participants with mild/moderate chronic plaque psoriasis. Methods: This was a randomized, double‐blind, first‐in‐human study (trial registration: ClinicalTrials.gov NCT03469336). Participants received each of the following six treatments once daily for 18 days: three topical doses (2.3%, 0.8%, 0.23%) of PF‐06763809, a vehicle and two active comparators (betamethasone and calcipotriol). Primary endpoints included change from baseline in psoriatic skin infiltrate thickness [echo‐poor band (EPB) on ultrasonography] at Day 19, and safety. Change in psoriasis‐associated gene expression (Day 19), evaluated by real‐time reverse transcription PCR of skin biopsies, was an exploratory endpoint. Results: In total, 17 participants completed the study. Change from baseline in the EPB on Day 19 for all three doses of PF‐06763809 was not significantly different from that of vehicle (P > 0.05). A significant reduction in EPB from baseline was observed with betamethasone on Day 19 relative to all other treatments (P < 0.0001). Treatment‐related adverse events were mild/moderate. There were no significant differences in gene expression on Day 19 between vehicle and PF‐06763809‐treated skin lesions. Conclusion: Using a psoriasis plaque test design, PF‐06763809 was found to be well tolerated with an acceptable safety profile in participants with psoriasis, but without reduction in skin infiltrate thickness or disease biomarkers. [ABSTRACT FROM AUTHOR]

The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6., (© 2024. The Author(s).)

Purpose: Accurate methods to determine dermal pharmacokinetics are important to increase the rate of clinical success in topical drug development. We investigated in an in vivo pig model whether the unbound drug concentration in the interstitial fluid as determined by dermal open flow microperfusion (dOFM) is a more reliable measure of dermal exposure compared to dermal biopsies for seven prescription or investigational drugs. In addition, we verified standard dOFM measurement using a recirculation approach and compared dosing frequencies (QD versus BID) and dose strengths (high versus low drug concentrations)., Methods: Domestic pigs were topically administered seven different drugs twice daily in two studies. On day 7, drug exposures in the dermis were assessed in two ways: (1) dOFM provided the total and unbound drug concentrations in dermal interstitial fluid, and (2) clean punch biopsies after heat separation provided the total concentrations in the upper and lower dermis., Results: dOFM showed sufficient intra-study precision to distinguish interstitial fluid concentrations between different drugs, dose frequencies and dose strengths, and had good reproducibility between studies. Biopsy concentrations showed much higher and more variable values. Standard dOFM measurements were consistent with values obtained with the recirculation approach., Conclusions: dOFM pig model is a robust and reproducible method to directly determine topical drug concentration in dermal interstitial fluid. Dermal biopsies were a less reliable measure of dermal exposure due to possible contributions from drug bound to tissue and drug associated with skin appendages., (© 2023. The Author(s).)

Objective: Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model., Methods: We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR., Results: RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P < 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (P < 0.05 in calcaneus, P < 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (γδ)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by RORγt inhibition (P < 0.001)., Conclusion: RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Plaque psoriasis (PsO) is an inflammatory skin disease driven, in part, by the activation of Janus kinase (JAK) signalling pathways., Objectives: To assess the efficacy and safety of multiple doses of topical brepocitinib, a tyrosine kinase 2/JAK1 inhibitor, in participants with mild-to-moderate PsO., Methods: This phase IIb multicentre randomized double-blind study was conducted in two stages. In stage 1, participants received one of eight treatments for 12 weeks: brepocitinib 0.1% once daily, 0.3% once or twice daily, 1.0% once or twice daily, 3.0% once daily, or vehicle once or twice daily. In stage 2, participants received brepocitinib 3.0% twice daily or vehicle twice daily. The primary endpoint was the change from baseline in Psoriasis Area and Severity Index (PASI) score at week 12, analysed using analysis of covariance. The key secondary endpoint was the proportion of participants who achieved a Physician Global Assessment response [score of clear (0) or almost clear (1) and an improvement of ≥ 2 points from baseline] at week 12. Additional secondary endpoints included the difference vs. vehicle in change from baseline in PASI, using mixed-model repeated measures, and the change from baseline in Peak Pruritus Numerical Rating Scale at week 12. Safety was monitored., Results: Overall, 344 participants were randomized. Topical brepocitinib did not result in statistically significant changes compared with respective vehicle controls in the primary or key secondary efficacy endpoints for any dose group. At week 12, least squares mean change from baseline in PASI score ranged from -1.4 to -2.4 for the brepocitinib once-daily groups vs. -1.6 for vehicle once daily, and from -2.5 to -3.0 for the brepocitinib twice-daily groups vs. -2.2 for vehicle twice daily. From week 8, change from baseline in PASI score separated from vehicle in all brepocitinib twice daily groups. Brepocitinib was well tolerated, with adverse events (AEs) occurring at similar rates across groups. One participant in the brepocitinib 1.0% once-daily group developed a treatment-related AE of herpes zoster in the neck area., Conclusions: Topical brepocitinib was well tolerated but did not result in statistically significant changes compared with vehicle when administered at the doses evaluated to treat signs and symptoms of mild-to-moderate PsO., Competing Interests: Conflicts of interest M.N.L. has served as investigator for AbbVie, Amgen, Galderma, Incyte, Novartis and Pfizer, and has served on an advisory board for Galderma. S.R.S. has been a consultant and member of the speaker’s bureau for AbbVie, Janssen and Lilly, and has been a clinical investigator for AbbVie, Aclaris, Arcutis, Galderma, Leo Pharmaceuticals, Lilly, Pfizer and Regeneron. G.B., G.F., P.G., G.F., V.P., S.A., C.B., E.P., M.S.V. and S.T. are employees of and shareholders in Pfizer Inc. J.S.B. was an employee of and shareholder in Pfizer Inc. at the time at which this study was conducted., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a β -arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [ 3 H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6 + peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases., (Copyright © 2023 by The Author(s).)

Macrocyclic retinoic acid receptor-related orphan receptor C2 (RORC2) inverse agonists have been designed with favorable properties for topical administration. Inspired by the unanticipated bound conformation of an acyclic sulfonamide-based RORC2 ligand from cocrystal structure analysis, macrocyclic linker connections between the halves of the molecule were explored. Further optimization of analogues was accomplished to maximize potency and refine physiochemical properties (MW, lipophilicity) best suited for topical application. Compound 14 demonstrated potent inhibition of interleukin-17A (IL-17A) production by human Th17 cells and in vitro permeation through healthy human skin achieving high total compound concentration in both skin epidermis and dermis layers., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

The application of machine learning to longitudinal gene-expression profiles has demonstrated potential to decrease the assessment gap, between biochemical determination and clinical manifestation, of a patient's response to treatment. Although psoriasis is a proven testing ground for treatment-response prediction using transcriptomic data from clinically accessible skin biopsies, these biopsies are expensive, invasive, and challenging to obtain from certain body areas. Response prediction from blood biochemical measurements could be a cheaper, less invasive predictive platform. Longitudinal profiles for 92 inflammatory and 65 cardiovascular disease proteins were measured from the blood of psoriasis patients at baseline, and 4-weeks, following tofacitinib (janus kinase-signal transducer and activator of transcription-inhibitor) or etanercept (tumor necrosis factor-inhibitor) treatment, and predictive models were developed by applying machine-learning techniques such as bagging and ensembles. This data driven approach developed predictive models able to accurately predict the 12-week clinical endpoint for psoriasis following tofacitinib (area under the receiver operating characteristic curve [auROC] = 78%), or etanercept (auROC = 71%) treatment in a validation dataset, revealing a robust predictive protein signature including well-established psoriasis markers such as IL-17A and IL-17C, highlighting potential for biologically meaningful and clinically useful response predictions using blood protein data. Although most blood classifiers were outperformed by simple models trained using Psoriasis Area Severity Index scores, performance might be enhanced in future studies by measuring a wider variety of proteins., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The pathogenesis of SpA is multifactorial and involves a range of immune cell types and cytokines, many of which utilize Janus kinase (JAK) pathways for signaling. In this review, we summarize the animal and pre-clinical data that have demonstrated the effects of JAK blockade on the underlying molecular mechanisms of SpA and provide a rationale for JAK inhibition for the treatment of SpA. We also review the available clinical trial data evaluating JAK inhibitors tofacitinib, baricitinib, peficitinib, filgotinib and upadacitinib in PsA, AS and related inflammatory diseases, which have demonstrated the efficacy of these agents across a range of SpA-associated disease manifestations. The available clinical trial data, supported by pre-clinical animal model studies demonstrate that JAK inhibition is a promising therapeutic strategy for the treatment of SpA and may offer the potential for improvements in multiple articular and extra-articular disease manifestations of PsA and AS.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Patients with psoriasis have an increased risk of myocardial infarction, and psoriasis is now recognized as an independent risk factor for coronary heart disease and cardiovascular mortality. To understand the effects of psoriasis medications on systemic inflammation associated with cardiovascular risks, we studied blood proteins related to inflammation and cardiovascular disease archived from a phase 3 clinical trial of tofacitinib and etanercept in adults with moderate-to-severe psoriasis. A total of 157 blood proteins were quantified by a proximity extension assay from 266 patients at baseline and week 4. Protein changes in the blood after 1 month of treatment were compared between tofacitinib (10 mg two times a day) and etanercept (50 mg biweekly), and by response status at week 12. Tofacitinib and etanercept commonly reduced IL-6, CCL20, and CXCL10, but IL-17A was significantly reduced only in responders of either treatment. Compared with etanercept, tofacitinib showed a wider spectrum of cardiovascular blood protein reduction, but the protein reduction effects of tofacitinib were strictly confined to treatment responders. Tumor necrosis factor receptor 1, E-selectin, hK11, tumor necrosis factor-related activation-induced cytokine, CHI3L1, IL-16, and matrix metalloproteinase-12 were cardiovascular proteins significantly reduced only in tofacitinib responders. Our data suggest that a short-term systemic psoriasis treatment can cause reductions in circulating inflammatory and other proteins associated with cardiovascular risks., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Interleukin 17 is a family of cytokines that play a central role in many autoimmune and inflammatory diseases. IL-17A has been implicated as a key driver of psoriasis, mediating a chronic cycle of T-cell activation, keratinocyte proliferation and angiogenesis. It has been hypothesized that expression of IL-17A and the related cytokine IL-17F could be used as predictive biomarkers for therapeutic response, though they have been difficult to measure locally or in circulation because of their low abundance. We developed ultrasensitive methods for measuring IL-17A and IL-17F in human serum samples and found that serum from psoriasis patients had higher and a broader range of concentrations of both IL-17 proteins compared to healthy volunteers. We also adapted these methods for tissue biopsies and saw higher concentrations of both IL-17 proteins in psoriatic lesions, but they were undetectable in non-lesional skin from the same patients.

The chemokine receptor CXCR5 is predominantly expressed on mature B cells and follicular T-helper cells. CXCR5 and its ligand CXCL13 participate in ectopic germinal center formation at the inflammatory sites of multiple immune diseases such as rheumatoid arthritis, multiple sclerosis, and Sjogren's syndrome. Therefore, disrupting CXCL13-induced chemotaxis may be a fruitful approach for developing therapeutics in treating these diseases. Cells undergo cytoskeletal rearrangement prior to chemotaxis, and therefore actin polymerization can be used as a surrogate readout more proximal to chemokine receptor activation than chemotaxis. Conventionally, actin polymerization is measured by fluorescence microscopy or flow cytometry, which are either of low throughput or in need of special instruments. We developed a 96-well actin polymerization assay that can process 1,000 to 1,500 samples a day. This assay uses a standard laboratory fluorescence microplate reader as the detection instrument and was optimized for various experimental conditions such as cell density, actin filament staining reagent, staining buffer, and cell culture conditions. We demonstrate that this actin polymerization assay in 96-well format exhibits the expected pharmacology for human CXCR5 and is suitable as a primary functional assay to screen neutralizing scFv in crude bacterial peri-preps and a secondary assay for small compound collections.

C5a is a terminal product of the complement cascade that activates and attracts inflammatory cells including granulocytes, mast cells and macrophages via a specific GPCR, the C5a receptor (C5aR). Inhibition of C5a/C5aR interaction has been shown to be efficacious in several animal models of autoimmune diseases, including RA, SLE and asthma. This account reports the discovery of a new class of C5aR antagonists through high-throughput screening. The lead compounds in this series are selective and block C5a binding, C5a-promoted calcium flux in human neutrophils with nanomolar potency., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Pre-stimulation of Chinese hamster ovary (CHO) cells expressing the human m1-muscarinic receptor (CHO-m1 cells) with a maximally effective concentration of the muscarinic agonist methacholine resulted in desensitization of Ins(1,4,5)P3 accumulation, apparent as a approximately 4-fold shift in the agonist dose-response curve. Agonist-induced desensitization was rapid (detectable by 10 s) and concentration dependent (EC50=8.2+/-2.2 microM) and resulted in a complete loss of receptor reserve for the agonist-stimulated Ins(1,4, 5)P3 response. An investigation of the possible mechanisms involved in m1-muscarinic receptor desensitization indicated that agonist-induced receptor internalization, PtdIns-(4,5)P2 depletion or an increased rate of Ins(1,4,5)P3 metabolism were not involved. m1-Muscarinic receptors did, however, undergo rapid agonist-induced phosphorylation with a time course that was consistent with an involvement in receptor desensitization. Characterization studies indicated that the receptor-specific kinase involved was distinct from protein kinase C and other second-messenger-dependent protein kinases. Since previous studies have suggested that the m3-muscarinic receptor subtype undergoes agonist-dependent phosphorylation via casein kinase 1alpha (CK1alpha) [Tobin, Totty, Sterlin and Nahorski (1997) J. Biol. Chem. 272, 20844-20849], we examined the ability of m1-muscarinic receptors to be phosphorylated by this kinase. In reconstitution experiments, CK1alpha was able to phosphorylate purified, soluble m1-muscarinic receptors in an agonist-dependent manner.

M1 muscarinic cholinergic receptors, G1 and G11 (Gq/11), and phospholipase C-beta 1 were highly purified from both natural sources and cells that express the appropriate cDNA's. When the proteins were co-reconstituted into phospholipid vesicles, the receptor efficiently and selectively promoted the activation of Gq/11, leading to marked stimulation of PLC activity in the presence of GTP gamma S. No stimulation was observed in the presence of GTP, however, which led to the finding that PLC-beta 1 stimulates the hydrolysis of GQ/11-bound GTP at least 50-fold. Thus, PLC-beta 1 is a GTPase activating protein, a GAP, for its physiologic regulator Gq/11. We discuss the implications of PLC-beta 1's GAP activity on the M1 muscarinic cholinergic signaling pathway.

Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.

The ability of several alkaloids to inhibit the metabolism and motility of human spermatozoa has been investigated. Of the agents tested, chloroquine was the most effective in inhibiting sperm metabolism (production of carbon dioxide and lactic acid) and motility. It was active at a concentration of 3.6 x 10(-5)M. Quinine and Quinacrine were active at concentrations of 5 x 10(-4)M and emetine required concentrations as high as 3.6 x 10(-3)M to achieve an inhibitory effect. Detailed studies with emetine showed that the time needed for inhibition of sperm motility was inversely proportional to the drug concentration and directly related to the sperm density. In addition, the inhibition was shown not to be reversible.

The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands.(ABSTRACT TRUNCATED AT 250 WORDS)