#	Aladdin ID	Assay Type	Description	Organism	Compounds	Reference	BAO Format	Source
1.	ALA3227482	A	Cytotoxicity against human astrocytes assessed as cell viability at 100 nM by AOPI staining-based fluorescence assay	Homo sapiens	1		cell-based format	Scientific Literature
2.	ALA3227483	B	Binding affinity to CCR5/MOR in human astrocytes assessed as inhibition of R5 HIV-1 SF162 infection by measuring Tat protein expression at 100 nM preincubated for 30 to 60 mins followed by viral infection measured after 18 hrs by luciferase reporter gene assay relative to maraviroc/naltrexone	Homo sapiens	1		cell-based format	Scientific Literature
3.	ALA3227484	B	Binding affinity to CCR5/MOR in human astrocytes assessed as inhibition of R5 HIV-1 SF162 infection by measuring Tat protein expression at 100 nM preincubated for 30 to 60 mins followed by viral infection measured after 18 hrs by luciferase reporter gene assay relative to maraviroc	Homo sapiens	1		cell-based format	Scientific Literature
4.	ALA3227487	F	Antagonist activity at CCR5 in Gqi5 transfected human MOLT4 cells assessed as inhibition of RANTES-stimulated Ca2+ influx preincubated for 15 mins followed by DAMGO challenge	Homo sapiens	6		cell-based format	Scientific Literature
5.	ALA3227488	B	Agonist activity at CCR5 in Gqi5 transfected human MOLT4 cells after 15 mins	Homo sapiens	1		cell-based format	Scientific Literature
6.	ALA3227489	B	Displacement of [125I]-MIP-1alpha from CCR5 in rhesus monkey Chem-1 cell membranes after 120 mins by liquid scintillation counting analysis	Macaca mulatta	4		cell-based format	Scientific Literature
7.	ALA3227490	F	Antagonist activity at human mu opioid receptor expressed in Gqi5 transfected CHO cells assessed as inhibition of DAMGO-stimulated Ca2+ influx preincubated for 15 mins followed by DAMGO challenge	Homo sapiens	3		cell-based format	Scientific Literature
8.	ALA3227798	B	Agonist activity at human mu opioid receptor expressed in Gqi5 transfected CHO cells after 15 mins	Homo sapiens	3		cell-based format	Scientific Literature
9.	ALA3227799	B	Displacement of [3H]-naloxone from human mu opioid receptor expressed in CHO cells after 1 hr	Homo sapiens	3		cell-based format	Scientific Literature
10.	ALA3706101	F	Flux Assay: As Ca2+ flux is associated with the activation of the MOR, the functional activity of bivalent ligand 1, monovalent ligand 2, and naltrexone was then evaluated in a Ca2+ mobilization assay in hMORCHO cells transfected with chimeric Ca2+ following a published protocol.23 No agonism was observed for any of the tested compounds (data not shown). Thus, they were further assessed for their antagonist properties as the ability to inhibit DAMGO (a MOR agonist) induced Ca2+ flux.	Homo sapiens	3		cell-based format	BindingDB Database
11.	ALA3706102	B	Radioligand Binding Assay: A bivalent ligand 1 (FIG. 14) that combines the pharmacophores of naltrexone (a MOR antagonist) and maraviroc (a CCR5 antagonist) into one molecule was designed and synthesized. Herein is reported the characterization of this novel molecular probe in for its binding affinity, Ca2− flux functional activity, and HIV-1 inhibition potency. Bivalent ligand 1 was first characterized in hMOR-expressed CHO cells in the competitive radioligand binding assay as described previously.	Homo sapiens	3		cell-based format	BindingDB Database
12.	ALA3706103	B	Radioligand Binding Assay: Afterwards, the pharmacological profile of bivalent ligand 1 at the chemokine receptor CCR5 was characterized similarly. The competitive radioligand binding assay was conducted in CCR5 rhesus macaque membrane preparations from Chem-1 cells. Monovalent ligand 3 and compound 4, an analogue of maraviroc, were tested along under the same condition. Introduction of the 4-NH2 group onto the phenyl ring of maraviroc, as seen in compound 4, caused approximately 65-fold decrease in the binding affinity, compared to maraviroc. The decrease of the binding affinity was even more profound for bivalent ligand 1 and monovalent ligand 3, as their Ki values dropped to submicromolar range, respectively.		4		cell membrane format	BindingDB Database
13.	ALA3706104	F	Flux Assay: Then the Ca2+ functional activity of bivalent ligand 1 was evaluated in the Gqi5 transfected CCR5-MOLT-4 cells as described in the literature.3 As expected, no CCR5 agonism was detected for the bivalent ligand 1 (data not shown). In the RANTES induced Ca2+ flux inhibition assay (Table 3), the bivalent ligand 1 was approximately 60-fold less potent than maraviroc. A more significant potency decrease (nearly 300 times) was observed for the monovalent ligand 3, compared to maraviroc. In order to figure out the possible reasons for such a dramatic drop of their potency, two analogues (4 and 5, FIG. 14) of mavaviroc carrying gradient steric hindrance characters at the same substitution position were evaluated under the same condition. Compound 4 showed a modest reduction of the potency (Table 2). However, the inhibition potency of the N-t-Boc protected analogue 5 dropped to micromolar (IC50=1.57+/-0.18 uM). It thus appeared that steric hindrance may play an essential role.		4		assay format	BindingDB Database