van Maanen MA , Papke RL , Koopman FA , Koepke J , Bevaart L , Clark R , Lamppu D , Elbaum D , LaRosa GJ , Tak PP , Vervoordeldonk MJ .

R01 GM057481 NIGMS NIH HHS , GM57481 NIGMS NIH HHS

	Figure 2. Compound activity of PMP-311 and PMP-072 on α7 nAChR.A, Competitive binding assay showed that PMP-311 and PMP-072 displaced the α7 nAChR-specific agonist α-bungarotoxin from binding to PC12 cells. B, PMP-311 showed a dose-dependent activation of α7 nAChR with a maximum about 85% that of ACh C, PMP-072 does not show to be an agonist of α7 nAChR ion channel activity. D, Voltage-clamp electrophysiological techniques revealed that application of PMP-311 (3 μM) to Xenopus oocytes expressing human α7 nAChR elicited a typical inward current. E, When PMP-072 was co-applied with 60 μM ACh to Xenopus oocytes voltage-clamped at 60 mV there was a concentration dependent decrease in the responses compared to ACh applied alone suggesting that with this experimental approach PMP-072 an antagonist of ACh-evoked α7 nAChR channel activation. In panels B & E each point represents the average of at least four cells (±SEM). Data were normalized to control responses to 300 μM ACh obtained prior to the application of PMP-311 (panel B) or 60 μM ACh applied without PMP-072 (panel E).
	Figure 3. Treatment with PMP-311 resulted in an amelioration of clinical signs of arthritis.Arthritis was induced in mice by immunization with type II collagen, and mice were treated with PMP-311 (n = 15, 5 mg/kg), PMP-072 (n = 17, 5 mg/kg), or saline (n = 15) by oral gavage from day 20 until day 34. A, Clinical score; Mice treated with PMP-311 showed a decrease in arthritis scores compared to saline-treated mice. B, Area under the curve (AUC) of the clinical score (day 20 to day 34) was decreased in PMP-311-treated mice versus control mice. C, Caliper score; Mice treated with PMP-311 showed a decrease in hind paw thickness, measured daily with a caliper, compared to the control group. D, AUC of the caliper score was decreased in PMP-311-treated mice compared with saline-treated mice. E, Disease incidence; PMP-311 reduced the incidence and delayed the onset of arthritis. * P < 0.05. compared to the control group.
	Figure 4. Inhibition of bone degradation and reduction of synovial inflammation in murine collagen-induced arthritis by PMP-311 (n = 15, 5 mg/kg).A, Semiquantitative scores of joint destruction. Joint destruction was decreased in mice treated with PMP-311. ** P < 0.01 compared to saline-treated mice B, Semiquantitative scores for synovial inflammation, assessed by hematoxylin and eosin staining of the knee joints, showed a decrease of synovitis in PMP-311-treated mice. * P < 0.05 compared to the control group.
	Figure 5. Dose-response study of PMP-311 and PMP-072 on clinical arthritis and paw swelling in murine collagen-induced arthritis.Mice were treated daily with PMP-311 (2, 5, 10 mg/kg, n = 15), PMP-072 (5, 10, 20 mg/kg, n = 15) or saline (n = 15) by oral gavage from day 20 until day 34. Percentages of areas under the curve (AUC) are shown. A, AUC of the clinical score was decreased more pronounced in PMP-311-treated mice versus control mice than in mice treated with PMP-072. B, Paw swelling was decreased in mice treated with PMP-311 at doses of 2 and 5 mg/kg and in mice treated with PMP-072 at 10 mg/kg. * P < 0.05 and ** P < 0.01 compared to saline treated mice.
	Figure 6. Inhibition of bone degradation and reduction of synovial inflammation in murine collagen-induced arthritis at different doses of PMP-311 (n = 15) and PMP-072 (n = 15) given by oral gavage from day 20 until day 34.A, Semiquantitative scores for radiographic joint destruction of the knee joints. Joint destruction was significantly decreased in mice treated with PMP-311 2 mg/kg and 10 mg/kg compared to the control group. B, Semiquantitative scores for synovial inflammation, assessed by hematoxylin and eosin staining of the knee joints, showed a decrease of synovitis in mice treated with PMP-311 2 mg/kg and PMP-072 10 mg/kg. * P < 0.05 and ** P < 0.01 versus saline-treated mice.
	Figure 7. Differential effects of PMP-311 and PMP-072 on human α7 nAChR in the resting state and following priming with the positive allosteric modulator PNU-120596.A, Initial responses to applications of 60 μM ACh (indicated by the bars) were obtained and then the α7-expressing cells were given a priming application of 300 μM PNU-120596 for 60 s (indicated by the bars over the third trace). No ion channel current was stimulated during the PNU-120596 application but subsequent responses to ACh were greatly increased in amplitude and duration. Note that 60 μM ACh was used for the control responses in this experiment since the effects of PNU-120596 priming on 300 μM ACh-evoked responses often resulted in responses that were too large to record under voltage-clamp conditions. B, Initial responses of α7 nAChR-expressing cells to applications of 60 μM ACh and 1 μM PMP-311 before and after priming with PNU-120596 (third trace). C, Initial responses of α7 nAChR-expressing cells to applications of 60 μM ACh and 10 μM PMP-072 before and after priming with PNU-120596 (third trace). D, Initial responses of α7 nAChR-expressing cells to applications of 60 μM ACh and the effect of an application of 10 μM methyllycaconitine (MLA) after priming with PNU-120596. In each panel five sequential 210 intervals of data are shown which were separated by 30s of additional wash (not shown). The bar graphs in each panel represent the average peak current responses of at least four oocytes (±SEM), normalized to the peak current of an initial 60 μM ACh-evoked response.
	Figure 1. Chemical structures of (A) PMP-311; (S)-2-(2-((Pyridin-3-yloxy)methyl)piperazin-1-yl)oxazolo[4,5-b]pyridine and (B) PMP-072; (R)-N-(4-methoxyphenyl)-2-((pyridin-3-yloxy)methyl)piperazine-1-carboxamide.

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