# US 10,767,212 B2 · Method of screening compounds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,212 B2 - Document ID: US10767212B2 - Status: Granted - Kind: B2 - Application number: 15/714,885 - Filed: 2017-09-25 - Published: 2018-04-12 - Granted: 2020-09-08 - Earliest claimed: 2016-09-23 - Assignees: Cornell University - Applicants: Cornell University - Inventors: Richard Cerione; Jon W Erickson; Clint A Stalnecker - Web page: https://patentgrep.com/patent/US10767212B2 ## CPC - C12Q 1/34: involving hydrolase - C12N 9/80: acting on amide bonds in linear amides {(3.5.1)} - C12Y 305/01002: Glutaminase (3.5.1.2) - G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) - G01N 2500/20: cell-free systems - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present technology relates to kidney-type glutaminase (GLS) proteins that have a phenylalanine to tryptophan substitution at the position corresponding to residue 322 of human glutaminase C (GAC), and methods of using such proteins to screen for compounds that bind to the activation loop of GLS and/or modulate glutaminase activity of GLS. ## Claims ### Claim 1 A method of screening for compounds that bind to the activation loop of kidney-type glutaminase (“GLS”) protein, said method comprising: contacting GLS protein, wherein the GLS protein comprises an activation loop and comprises a phenylalanine to tryptophan substitution at the residue corresponding to position 322 of SEQ ID NO: 2, with a test compound, wherein the test compound is a compound that potentially binds to the activation loop, to permit binding between the activation loop and the test compound; detecting fluorescence of the substituted tryptophan in the presence of the test compound; comparing the detected fluorescence to fluorescence of a control; and identifying, based on said comparing, that the test compound is a compound that binds to the activation loop of GLS, wherein a change in fluorescence relative to the control indicates that the test compound binds to the activation loop. ### Claim 2 (depends on 1) The method according to claim 1, wherein said contacting is carried out in vitro. ### Claim 3 (depends on 1) The method according to claim 1, wherein said contacting comprises incubating the protein with the test compound for ten to thirty minutes. ### Claim 4 (depends on 1) The method according to claim 1, wherein the test compound is selected from the group consisting of bis-thiadiazoles, small inorganic anions, and negatively-charged metabolites. ### Claim 5 (depends on 1) The method according to claim 1, wherein said comparing comprises: (a) calculating the binding affinity between the test compound and the protein from the detected fluorescence and (b) comparing the calculated binding affinity to that of the control. ### Claim 6 (depends on 1) The method according to claim 1, wherein the fluorescence of the control is the fluorescence of the substituted tryptophan in the absence of the test compound. ### Claim 7 (depends on 1) The method according to claim 1, wherein the control is a second kidney-type glutaminase (“GLS”) protein comprising a phenylalanine to tryptophan substitution at the residue corresponding to position 322 of SEQ ID NO: 2 and the fluorescence of the control is the fluorescence of the substituted tryptophan of the second GLS protein detected in the absence of the test compound. ### Claim 8 (depends on 1) The method according to claim 1 further comprising: contacting kidney-type glutaminase (“GLS”) protein with glutamine in the presence of the test compound under conditions effective for the protein, if active, to convert glutamine to glutamate; measuring production of glutamate, if any, following said contacting with glutamine; comparing the measured glutamate production to glutamate production of a second control; and further identifying, based on said comparing glutamate production, that the test compound is a modulator of glutaminase activity of GLS, wherein a change in glutamate production relative to the second control indicates that the test compound modulates glutaminase activity of GLS. ### Claim 9 (depends on 1) The method according to claim 1 further comprising identifying a compound that also modulates glutaminase activity of kidney-type glutaminase (“GLS”) protein, said further method comprising: selecting a test compound identified as being a compound that binds to the activation loop according to said identifying step; contacting GLS protein with glutamine in the presence of the selected test compound under conditions effective for the protein, if active, to convert glutamine to glutamate; measuring production of glutamate, if any, following said contacting with glutamine; comparing the measured glutamate production to glutamate production of a second control; and further identifying, based on said comparing glutamate production, that the selected test compound is also a modulator of glutaminase activity of GLS, wherein a change in glutamate production relative to the second control indicates that the selected test compound also modulates glutaminase activity of GLS.