See the: Virtual ToDo List

Includes:

- Biotin:Avidin docking comparison

- ICM vs GOLD vs. VINA

- Docking Comparison: Rosie (Rosetta) vs. Drug Discovery at TACC (Vina)

do most inhibitors of metallo enzymes try to displace the metals? Or do they bind with the metals present?

102815

List of other labs that studens may go to:

Deprecated Projects:

Wolbachia Targets

SRC Project

052815

Use Rosetta Ligand Docking server with advanced students to compare against GOLD/ICM/VINA

http://rosie.rosettacommons.org/ligand_docking/submit

Use PyRosetta or FOLDIt GUI interfaces for Rosetta to do loop modeling/ - maybe homology models.

052215 - Walt won $750 faculty resaerch grant for buying inhibitors of NDM-1 to test.

We have to spend this money in 3 months!

052115- Target ideas from Luis

Luis says - his Burkholderia pseudomallei target is a shunt of the glycoxylate pathway which is itself a shunt to the sugar metabolism pathway

He recommends: Isocytrate lyase & malate synthase - are actually both good targets within the glyoxylate pathway

metabolize fatty acids instead of sugars

Pathogenic bacteria and fungi

Good in Tuberculosis and Candida albicans

Is there a Isocytrate lysas or malate synthase in Burkholderia ?

051815 - John G to download and print some of these posters - and hang in the lab

http://cbc.arizona.edu/njardarson/group/top-pharmaceuticals-poster

2015 Summer students to work on 3-D printing

- each makes one 'thing'

- teach mentors first

051815

DSF

- on NDM-1 using EDTA (Does removing metals destabilize the protein) - Oscar did this

- on NDM-1 with inhibitors - Oscar did this with L-captopril

- on RAS (does fluorogenic probe destabilize RAS - which may explain RAS protein degradation in cells)

051315

Targets that Walt would like us to do:

metallo-beta-lactamase and some serine-beta-lactamase targets

"Between a collaborator and I, we have expression plasmids for metal-dependent (NDM-1, IMP-7 (similar to structure of IMP-1), VIM-2) beta lactamases and a serine beta lactamase (KPC-2). And we could get others in each category. They are not currently His-tagged, but could serve as templates for PCR.

I said we can order the Oligo primers with His tag and make them in house - but would be good to have the plasmid (non-HIS) as backups.

IMP-7: (metal-dependent)

pET-28b

His Tag

Sequence attached

VIM-2: (metal-dependent)

pET-24a

No His Tag

KPC-2: (serine beta lactamase)

pET-24a

His Tag

ToDo:

- sequence verify these:

IMP-7: (metal-dependent), pET-28b, His Tag

VIM-2: (metal-dependent), pET-24a - No His Tag

KPC-2: (serine beta lactamase), pET-24a, His Tag

Re-clone this one into pNIC-Bsa4 to get HIS Tag:

VIM-2: (metal-dependent), pET-24a - No His Tag

NOTES ON IMP7 vs. IMP1:

We were originally going to go with IMP-1, but Crowder (who sent the plasmid) tells me that IMP1 crashes out of solution a lot, and they have had better luck with IMP-7, so he sent IMP-7 instead.

There are structures for IMP1, but not for IMP7. they appear to be about 90% identical.

We could dock to IMP-1 (or a homology model of IMP7) and then use IMP7 for the wet lab work.

I have not yet mapped where the substitutions occur. If it is a problem, I can always go back to Mike and ask him for IMP-1.

ToDo:

Map where the changes between IMP7 and IMP1 are using PyMol.

Make IMP-7 homology model on ICM

Dock against IMP1 structure

Dock against IMP7 homology structure

ENTER THE LINKS TO YOUR TARGET PAGES HERE:

6-phosphoglucanate dehydrogenase (L. monocytogenes)...................................................Researcher names

6-phosphoglucanate dehydrogenase (T. gondii)...................................................................Researcher names

Hypoxanthine phosphoribosyltransferase (P. falciparum)......................................................Researcher names

Histidinol-phosphatase (M. tuberculosis)...............................................................................Researcher names

Glucose-1-phosphatase (S. enterica)....................................................................................Researcher names

D-alanine ligase (K. pneumoniae).........................................................................................Researcher names

S-adeosylmethionine syntheses (L. major)...........................................................................Researcher names

S-adeosylmethionine syntheses (P. falciparum)....................................................................Researcher names

S-adeosylmethionine syntheses (T. brucei)...........................................................................Researcher names

S-adeosylmethionine syntheses (T. gondii)...........................................................................Researcher names

0825/16 - Previous Researcher's target page here - we may have a new set of students work on this for Fall 16

TargetF15 - S-adenosylmethionine Synthetase (Mycobacterium tuberuclosis)....................................Bethany R., Ana R.

TargetF15 - D-alanine Ligase (Listeria monocytogenes).....................................................................Simone M.

TargetF15 - 6-phosphoglucanate dehydrogenase, decarboxylating, putative (Trypanosoma cruzi)...Diane C.

TargetF15- 6-phosphogluconate dehydrogenase, decarboxylating, putative (Leishmania major)....Mayur P.

TargetF15 - S-adenosylmethionine Synthetase (Burkholderia pseudomallei)......................................Matthew N., Justin H.

Fall 2015 Targets

Primer Design for Springers: (09/21/15)

Oligo Primers:

ProtocolPCR_PrimerDesign_OverlapAssemblyVDS_Summ15 in the GDrive/VDSclass/Protocols/FirstProtocols

Tail Primers

ProtocolPCR_PrimerDesignTails_for_pNIC-Bsa4CloningSumm15.doc in the GDrive/VDSclass/Protocols/CloningProtocols

TargetF15- Inosine 5 Monophosphate Dehydrogenase (Bacillus anthracis).......................................Ashley C., Lisa S.

TargetF15 - Adenylosuccinate Synthetase (Plasmodium falciparum).................................................Kevin H.

TargetF15 - D-alanine Ligase (Staphylococcus aureus)......................................................................Kevin N., Anthony A.

TargetF15 - Protein Tyrosine Phosphatase (Listeria monocytogenes)..................................................Elizabeth K. Krupa S. Sabryna S.

TargetF15 - Serine Threonine Protein Phosphatase 1 (Plasmodium falciparum)...................................Kamia R., Jonathan W., Elizabeth M., Neethu G.

TargetF15 - Secreted Phosphatase (Francisella tularensis) ................................................................Juliana Z., Samantha V., Mahin D

TargetF15 - Protein Phosphatase 2A, Putative (Trypanosoma brucei)..............................................Kylie J., Marisa S., Margot F., Cindy N.

TargetF15- Inorganic Pyrophosphatase (Rickettsia prowazekii)............................................................Ariella S., Brittan M., Andre P.

Other Databases to look into for targets:

http://omictools.com/certain-infectious-and-parasitic-diseases-c1354-p1.html

Try TuberQ & Tuberculist

Fall 2014 Targets (Springers)

Target - Phosphoserine phosphatase (Vibrio cholerae).............................................John G., Keenan W., Samantha D., Avery W.

Mentor: Oscar and Nicole

Target - Serine Threonine Phosphatase STP1, (Streptococcus Agalactiae) ...............Jairo V, Saniya H, Stacy T, Daren N

Mentor: Kavya and Melissa

Target - 6-phosphogluconate dehydrogenase, decarboxylating, putative (P. falciparum 3D7) .....................Saul H, Lauren M, Carlos Gio O.

Mentor: Vicky and Serena

Target - Secreted Phosphatase (Mycobacterium tuberculosis)................................. Justin S., Jazmine A., Anh P., Alberto T.

Mentor: Priya and Anita

Target-Protein Tyrosine Phosphatase (Listeria monocytogenes)................................Monica L., Tyler T., James F.

Mentor: Grace

Summer 2014 Targets (Vets)

Target - Pteridine Reductase 1, (L. Major)................................................................Andrei L., Hailey D.

Target - D-alanine:D-alanine Ligase. (M. tuberculosis)...............................................Charina B., Chris T.

Target - 6-phosphoglucanate dehydrogenase (P. vivax).............................................Nikki W., Carolyn T.

Target-protein tyrosine phosphatase (Entamoeba histolytica)....................................Andee F. (Vicky G.) - ARISp16?

Target - Secreted Phosphatase (Francisella tularensis).............................................Cidia G.

Target - acetoacetyl-CoA reductase - Burkholderia pseudomallei................................Luis V., Zain A.

Target - GES-5 carbapenemase (K. pneumoniae) .................................................................................................Xenia G.

Summer 2015 Target Discovery

Part Uno

With a partner, select a target from the TDR Targets exercise to evaluate.

https://docs.google.com/spreadsheets/d/1etngvvFh0LIAGP13vQcgE9CxWjxjQjuxNNFEVzSMDAs/edit?usp=sharing

Put your names in the NOTES A section. (First name Last Initial e.g. Spongebob S.)

Determine why it should be keep in consideration or not

If NO

• • write notes in the 'NOTES A' field

  • Color that notes cell RED

  • Then move the line to the 'Trash' sheet

If YES

• • write notes in the 'NOTES A' field

  • Color that notes cell GREEN

  • leave the line on that page

If MAYBE

• • write notes in the 'NOTES A' field

  • Color that notes cell YELLOW

  • leave the line on that page

Once finished - repeat for another target.

Part Dos

As individuals, select one of the Targets from the Spring 2015 Target Discovery on Wikispaces (see below)

Choose one that is NOT yours.

Then create a new line in the Targets_VDSSumm15

https://docs.google.com/spreadsheets/d/1dymtoVCnFwbX-kOaDWTyWh7KrQ5OZXLw5voZP9xaPb0/edit?usp=sharing

Rinse and repeat until we have been through them all.

Part Tres

Otros Objetivos

Dr. B selections, etc

Luis's recommendations

Step A: finish going through Wiki targets and adding them to Targets_VDSSumm15

Step B: check TDRTargetsExercises for verify hits on Main sheet

Step C: Transfer top TDR Targets to Targets_VDSSumm15

Step D: Search and enter info for the below Additional Target into Targets_VDSSumm15

Additional Targets (from Dr .B)

Targets from Prior Years Discovery Sessions that were not used:

6-phosphogluconate dehydrogenase - (in other than T. brucei or P. vivax) --> P. falciparum 3D7, or Leishmania major Friedlin strain

1.1.1.49

D-alanine:D-alanine ligase (in other than M. tuberculosis)

3-oxoacyl-(acyl-carrier protein) reductase, putative (P. aeruginosa)

3-oxoacyl-(acyl-carrier protein) reductase, L. major strain Friedlin LmOACP

1.1.1.10

Inosine-5'-monophosphate dehydrogenase EC 1.1.1.205 - not that essential ?

Glutathione reductases in any infectious organism

1.6.4.2 and 1.8.1.7

Thiordoxin Reductase in any infectious organism

- P. falciparum 3D7

T. brucei Tb927.3.1240 OG4_10586 protein phosphatase 2A, putative

4EM8 Anaplasma AnphA.00455.a ribose 5-phosphate isomerase B

Pfal008434AAA PFE0730c ribose 5-phosphate epimerase (isomerase), putative yes Pentose phosphate pathway should be essential?

3QH4 Mycobacterium MymaA.00277.c Esterase LipW - PNPA assay

Phosphatase in T. cruzi

http://tdrtargets.org/targets/view?gene_id=34880

View the highlighted targets in this GDrive sheet: (Seattle Targets) - there are about 5-7 of these

SSGCIDstructures_noassocpub_1

Spring 2015 Target Discovery

PART 1:

TDR Targets Searches Exercise (for practice)

PURPOSE:

find genes for these criteria using the TDR Targets webpage and then enter them onto spreadsheet:

• Pick a search, Perform the search, See which targets are found in the list.

• If the target is already on the spreadsheet - go to the next one that hasn't been entered yet.

  • Put your name down in the 'SOURCE / PI' column for that target and fill in the rest of the information

Phosphatases in T. brucei that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in T. cruzi that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in P. falciparum hat have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in P. vivax that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in T. gondii that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in M. tuberculosis that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in L. major that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Reductase in all (except C. elegans) that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.6 (be sure to search for reductase and check the box for enzyme)

Spreadsheet for TDR Target

https://docs.google.com/spreadsheets/d/1etngvvFh0LIAGP13vQcgE9CxWjxjQjuxNNFEVzSMDAs/edit?usp=sharing

PART 2:

SELECTING YOUR TARGET

NOTE: do this ONLY after you have completed the above TDR Targets exercise.

Follow the rest of the Target Discovery Protocol and create a new Wikispaces page that has all the info you need for a target. (the Wikispaces page will link from here)

Spring 2015 Target Selection Pages

ENTER THE LINKS TO YOUR TARGET PAGES HERE

*NOTE; Do not use "/" in your page titles

"Target - <protein name> <species> ................<your name(s)>"

How To Create a New Target page

Login to Wikispaces, Edit this Targets page with a title that will link to your new page, Type a new line that says:

TargetSp15 - Disulfide Oxidoreductase (Burkholderia Pseudomallei).....................Researcher: Brittan M.

TargetSp15- Acetylglutamate kinase (Rickettsia rickettsii)...............Researcher: Michael S.

Target Sp15 - Glucose-1-Phosphatase (Salmonella Enterica).....................Researcher: Kamia R.

TargetSp15- Phosphoglycerate kinase (Trypanosoma brucei)..................Researcher: Margot F.

TargetSp15-BETA-KETOACYL-ACP SYNTHASE III (Plasmodium Falciparum).........................Researcher: Bethany R.

TargetSp15- 2-dehydro-3-deoxyphosphooctonate aldolase (Burkholderia pseudomallei).................Researcher: Ashley C.

TargetSp15 - 2-acetamido-2-deoxy-alpha-D-glucopyranosyl-(1->6)-phosphatidylinositol de-N-acetylase (Trypanosoma brucei)..... Researcher: Cindy N.

TargetSp15- Triosephosphate Isomerase (Trypanosoma brucei)............Researchers: Neethu G.

TargetSp15- Serine threonine-protein phosphatase (Plasmodium falciparum)............ Researchers: Ariella S.

TargetSp15-hypoxanthine phosphoribosyltransferas (Plasmodium falciparum 3D7)......... Researchers: Lisa S.

TargetSp15- Serine/Threonine-Protein Kinase B PKNB (Mycobacterium tuberculosis).......Researchers: Emily B.

TargetSp15 - Trehalose-6-Phosphate Phosphatase (Aspergillus fumigatus)................Researchers: Andre P. (this page doesn't exist)

TargetSp15 - Thioredoxin Reductase (Listeria monocytogenes)...............Researchers: Anthony A.

TargetSp15 - N-Acetyl-gamma-Glutamyl-Phosphate-Reductase (Mycobacterium tuberculosis)...............Researchers: Mayur P.

TargetSp15 - Thioredoxin Reductase (Mycobacterium tuberculosis)................... Researchers: Jeffrey X.

TargetSp15 - protein-tyrosine-phosphatase A (ptpA)................Researchers: Karan K.

TargetSp15 - Isoleucine-tRNA Synthetase, Putative (Toxoplasma gondii)...............Researchers: Bianca E.

TargetSp15 - Phosphotyrosine protein phosphatase (Mycobacterium tuberculosis)......................Researchers: Simone M.

Target Sp15 - Acetyl-CoA carboxylase (Clostridium botulinum)

...............Researchers: Juliana Z.

Target Sp15 - Adenylosuccinate synthetase (P. falciparum)...............Researchers: Elizabeth K

TargetSp15 - Inositol Monophosphatase (Mycobacterium tuberculosis)...........Researcher: Steven T.

TargetSp15 - Fab V Enoyl-ACP Reductase (Yersenia Pestis)..................Researchers: Mahin D.

TargetSp15 -NADH-depemdent Enoyl-(acyl-carrier-protein) reductase (Mycobacterium tuberculosis)............ Researcher: Krupa S.

TargetSp15 - Glucosyl-3-phosphoglycerate phosphatase (Mycobacterium tuberculosis)..............Researcher: Sabryna S.

Target Sp-15- HPPK-DHPS, 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Francisella Tularensis) ……..Researcher: Kylie J

TargetSp15 - Histone deacetylase (Plasmodium berghei) ....................................................... Researcher: Jonathan Wang

TargetSp15 - NucleosideTriphosphateDiphosphohydrolase (Toxoplasma gondii)..........................Researcher: Kevin H.

TargetSp15 - Penicillin-binding protein 2x (Streptococcus Pneumoniae)..................................Researcher: Kevin N.

TargetSp15 - Adenylate Kinase (Leishmania major) ............................Researcher: Samantha Vargas

TargetSp 15 - E3 Ubiquitin-protein ligase (Salmonella Typhimurium)..............................................Researcher: Matthew N.

TargetSp15 - Acetate Kinase (Mycobacterium Paratuberculosis)................................................Researcher: Diane C.

TargetSp15 - CAI-1autoinducer synthase (Vibrio Cholerae).............................................................Researcher: Mina C.

TargetSp15 - ATP-dependent zinc metalloprotease (Bacillus Anthracis)...................................Researcher: Justin H.

TargetSp15 - Acidocalcisomal exopolyphosphatase (Leishmania major ).....................Researcher - Edward S.

For Example:

TargetSp15 - Enzyme Name (Organism name)........................................................Researchers: Dr. B

TargetSp13 - Fab G 3-ketoacyl-(acyl-carrier-protein) reductase.(Rickettsia prowazeki

i)...................................................................Researchers: Priya P. Oscar V Betty H - - ARISp16?

040115

Pulled information off of target page so that Spring15 studetns can start fresh:

Summer 2014 Target Discovery

Pull best Targets from the

• 1. TDR Target Discovery Exercise

  2. Individual's Target pages

  3. The SSGCIDstructures_noassocpub_1 (Seattle Structural Genomics)

     1. • note: these are ones that have PDB structures but no publications !

  4. Past searches from 2013 (see Targets/Archive folder

  5. New searches

Small groups discuss, compare their top finds.

Select 5 targets for the group to work on this summer

Do we want to use any ongoing targets? (YopH, FtHap, RpFabG)

Tbrucei 6PG - robust assay

Post info to TargetSelections_VDSSumm15 on Google Drive

Link to Target Selection Form: Spring 2015

https://docs.google.com/spreadsheet/viewform?formkey=dDgtRkMzVGp1UzYwWHZlMlplNk9XZUE6MA

Spring 2014 Target Discovery

Note from Dr. B.

-When searching for structure for Part 1, see bottom of page 4 of the Target Discovery Protocol; begins with "If no Xray crystal structure..."

Be sure to search against the PDB database.

Note: When getting amino acid sequence for BlastP, link out from TDR targets to other database and scroll to the bottom.

PART 1:

TDR Targets Searches Exercise (for practice)

PURPOSE:

find genes for these criteria using the TDR Targets webpage and then enter them onto spreadsheet:

• Pick a search, Perform the search, See which targets are found in the list.

• If the target is already on the spreadsheet - go to the next one that hasn't been entered yet.

  • Put your name down in the 'SOURCE / PI' column for that target and fill in the rest of the information

Phosphatases in T. brucei that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in T. cruzi that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in P. falciparum hat have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in P. vivax that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in T. gondii that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in M. tuberculosis that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Phosphatases in L. major that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.5.

Reductase in all (except C. elegans) that have no transmembrane regions, have essentiality evidence, and are druggable at least to 0.6 (be sure to search for reductase and check the box for enzyme)

Spreadsheet for TDR Target

https://docs.google.com/spreadsheets/d/1etngvvFh0LIAGP13vQcgE9CxWjxjQjuxNNFEVzSMDAs/edit?usp=sharing

PART 2:

SELECTING YOUR TARGET

NOTE: do this ONLY after you have completed the above TDR Targets exercise.

Follow the rest of the Target Discovery Protocol and create a new Wikispaces page that has all the info you need for a target. (the Wikispaces page will link from here)

Spring 2014 Target Selection Pages

ENTER THE LINKS TO YOUR TARGET PAGES HERE

*NOTE; Do not use "/" in your page titles

Target - Acidocalcisomal Exopolyphosphatase. Leishmania major.........................................................................Ashly O.

Target- PHOSPHOTYROSINE PROTEIN PHOSPHATASE PTPB (Mycobacterium tuberculosis) ............................Ariana Lerma

Target-serine-threonine protein phosphatase. (P. falciparum 3D7)...........................................................................Lauren McGregor - PDB off

Target- protein-tyrosine-phosphotase ywlE ..........................................................................................................Taylor Castillo

Target - Aspartate Aminotransferase. Leishmania major (Friedlin strain) ................................................................James F

Target - Trypanosoma brucei, protein tyrosine phosphatase ..................................................................................Researcher: Keenan W.

Target- Glutamine Synthetase (Mycobacterium tuberculosis) ............. Researcher: Justin S.

Target - GES-5 carbapenemase (K. pneumoniae) .................................................................................................Xenia G.

Target - Calcium Bound LIPL32 (Pathogenic Leptospira).........................................................................................Sara C.

Target - Protein phosphatase-beta, putative (P. vivax)............................................................................................Carolyn Trieu

Target - Tyrosine-protein phosphatase yopH...........................................................................................................Daren N.

Target - glutathione reductase...............................................................................................................................David Ton

Traget- tyrosine hydroxylase (T. gondii Toxoplasmosis)...........Saul H.

Target - Quiescin sulfhydryl oxidase - Trypanosoma brucei.....................................................................................Tyler Tschirhart

Target - dual specificity phosphatase, catalytic domain-containing protein (Toxoplasma gondii)..................................Anh Pham

Target - UDP-N-acetylglucosamine pyrophosphorylase (Aspergillus fumigatus).........................................................Anna Bozhkova

Target - S-adenosylhomocysteine hydrolase (L. major)............................................................................................Andee F.

Target - Probable Adenosylhomocysteinase SAHH (S-Adenosyl-L-Homocysteine Hydrolase) (Adohcyase) (Mycobacterium tuberculosis)......Cidia G.

Target - Tyrosyl-tRNA synthetase (L. major)...............................Researcher: Monica L.

Target- DIHYDROLIPOAMIDE DEHYDROGENASE LPD (LIPOAMIDE REDUCTASE (NADH)) (Mycobacterium tuberculosis)....Samantha D.

Target - Probable phosphoribosyl-AMP pyrophosphate HisE (Mycobacterium tuberculosis)................................................................Sanjna Z.

Target - Island Protein C (IglC) Francisella tularensis ………...................................................................................Avery W

Target - Serine-threonine specific protein phosphatase (//Taxoplasma Gondii)// .......................................................Jazmine Alanis (genus spelling)

Target - serine-threonine protein phosphatase 2b (catalytic subunit), putative (Plasmodium vivax)...................Saniya Hirani

Target - UDP-N-acetylbacillosamine N-acetyltransferase (Campylobacter jejuni)………………………............Charina Benavidez

Target - Succinate Dehydrogenase (Flavoprotein subunit) SDHA (Succinic dehydrogenase) (Fumarate reductase) (Fumarate dehydrogena).....Nikki Weber

Target - Malate Dehydrogenase (L. major, Friedlin Strain) ........................................................................................Andrei Loghin

Target- Cyclooxygenase-2 (Francisella tularensis)....................................................................................................John G.

Target - acetoacetyl-CoA reductase - Burkholderia pseudomallei ..............................................................................Luis Valencia

Target - Enoyl-acyl-carrier-protein reductase //Plasmodium falcifarum 3D7//...............................................................Researcher: Stacy Tunggadjaja

Target - deoxyuridine triphosphatase (Leishmania major)..........................................................................................Alberto T.

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

Beginning of Fall 2014

Pulled Target pages - so that students can re-start them on their own:

Were done originally in 2012, then in 2013 Fall, now re-using for 2014 Fall Springers

Target - S. agalactiae serine-threonine phosphatase STP1 ---------------- [NOT CLONED LAST YEAR]+page Researcher: Brandy C. Caroline C, D'Ondria P

JANICE

Target - Protein Tyrosine Phosphatase (Listeria monocytogenes) ------ [NOT CLONED LAST YEAR] - low homology Researcher: James T, Jesus D, Jiaqi Z

BRANDON

Target- phosphoserine phosphatase (Vibrio cholerae) -----------------------------------------(Created by William E.) Researcher: Dax F.G., Will E., Julia C.

SUMAN

Target- Protein phosphatase 2C (Toxoplasma Gondii) -----------------------------------(Created by Jensen G.) Researcher:Jensen G, Imran Z

HERE they are under the 2012 banner:

2012 Targets

Targets that were not successfully cloned in 2012

Target-serine-threonine protein phosphatase (Leishmania major)-------------------------------------- Paul, Ling, Rishi [NOT SUCCESSFULLY CLONED]

Target- Protein Tyrosine Phosphate (Yersinia Enterocolitica).......................................... Ivy, Stephanie, Jennifer [NOT SUCCESSFULLY CLONED]

Target- Protein Phosphatase 2B................................................................................... Divya, Tom, Sajan [NOT SUCCESSFULLY CLONED]

Target - S. agalactiae serine-threonine phosphatase STP1 ------------------- Suman, Brandon, Ruifei [NOT SUCCESSFULLY CLONED]

Target - Protein Tyrosine Phosphatase (Listeria monocytogenes) ------- Akhilesh, Mihir, Shane [NOT SUCCESSFULLY CLONED]

Fall 2013

Spreadsheet Target Selections for Fall 13

As static webpage:

As spreadsheet you can edit:

Targets that we have Oligos for but have not started cloning:

Target - Alpha Carbonic Anhydrase (Helicobacter pylori) ..................................................(Created by Marianna U.) Researcher: Jessica N, Qasim A, Kelly

DANIEL

Target - serine/threonine protein phosphatase catalytic subunit, putative (//T. cruzi//) ...........(Created by Priya) Researcher: Grant T, Aditi S.

ANDREW

Target -Serine, threonine protein phosphatase 2b (catalytic subunit), putative (P. viva)...........(Created by Feng Gao) Researcher: Anita V, Ariel C, Gautam

MAX

Targets that were not successfully cloned in 2012 (We have Oligos for some of these - but will order fresh oligos).

Target-serine-threonine protein phosphatase (Leishmania major)------------------------- [NOT CLONED LAST YEAR] Researcher: Fin G, Daniel D, Manuel Z

MICHAEL, ALDO

Target- Protein Phosphatase 2B.(T. brucei)...................................................... [NOT CLONED LAST YEAR] -page Researcher: Keely W, Nicole W

URVASHI, DIVYA

Researcher: Brandy C. Caroline C, D'Ondria P

JANICE

Researcher: James T, Jesus D, Jiaqi Z

BRANDON

Researcher: Dax F.G., Will E., Julia C.

SUMAN

Researcher:Jensen G, Imran Z

MAX

Summer 2013 Targets that are now being worked on in Fall

Spreadsheet Target Selections for Summer 13

https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodHRXZGxpZTJjMWl0UXN2STYzcmx2R3c&usp=sharing

Codon Frequency Table for E. coli K-12 (class 2)

Links to Individual Target Pages

"Target - Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) [Leishmania Major]--------------------------------Researchers: Tony Garcia, Renée French

JANICE

Target - FabG 3-oxoacyl-(acyl-carrier protein) reductase (P. falciparum) .......................... Researchers: Melissa H. & Jason P. -

- ARISp16?

DANIEL

Target- 3D7, glutathione reductase......................................................................................(Created by Seo Kelly K.) Researchers: Alyssa K. and Stephanie B.

BRANDON

Target - Serine threonine protein phosphatase PP1 (Plasmodium falciparum)..........................(Created by Anita V.) Researchers: Katherine V., Marianna U., and Ashlee W.

MICHAEL, ALDO

Target - Fab G 3-ketoacyl-(acyl-carrier-protein) reductase.(Rickettsia prowazekii)...................................................................Researchers: Priya P. Oscar V Betty H

URVASHI, DIVYA

Target_Serine_threonine_protein_phosphatase5_(Trypanosoma brucei)............................... Researcher: Ramiro R.

ANDREW

Target - β-carbonic anhydrases (Mycobacterium tuberculosis) ......................................... (Created by Ashlee W.) Researcher: Nicolet F.

ANDREW

Target- 6-phosphogluconate dehydrogenase (T. brucei).............................................................(Created by Emily J.) Researchers: Kavya K. and Vicky G. - ARISp16?

MAX

Target - Protein Phosphatase 2A, putative (T. Brucei) ............................................................................Researcher: Jacky E & Kevin E

SUMAN

Target - 3-oxoacyl- (acyl-carrier-protein) reductase (Mycobacterium tuberculosis)............ (Created by Caroline C) Researcher: Grace & Young - ARISp16?

ANDREW

Target-New Delhi Metallo Beta Lactamase .........................................................(Created by Madeline J.) Researchers: Serena Z., Madeline J., Brendan C.

URVASHI, DIVYA

Seattle Structural Genomics Targets - see my Targets folder.

Anaplasma, Babesia, Bartonella, Borrelia, Brucella, Burkholderia, Coccidioides, Cryptosporidium, Ehrlichia, Encephalitozoon, Entamoeba, Giardia, Mycobacterium, Rickettsia, Salmonella

Fall 2013

PseudoKnot Project

ProMol Project

Celiac Project

Target Selection Form

NOTE: do this AFTER looking at the Targets above

https://docs.google.com/spreadsheet/viewform?formkey=dEFwQWp6M2lPZFRkcl9HcjBrT1lTOXc6MA#gid=0

Target Assignments for Springers

https://docs.google.com/spreadsheet/pub?key=0AvGl3lMyhDsodDZtbjJSSzVITHNuXzlIOVQwYThWRXc&output=html

092213

To see what items were missing from your Target page for your grade, see this spreadsheet (it is also in the GDocs/Mis/Targets folder)

https://docs.google.com/spreadsheet/pub?key=0AvGl3lMyhDsodE1kWEFTc2xsTnRLOGhvdE54RGNaVWc&output=html

Interesting Ones - but nor pursued for some reason:

Target-Histidinol Phosphatase (Escherichia coli O157 H7)---------------------------------------------(Created by Min S.) - wait on this one. Not sure of essentiality of assay.

Target:Inositol phosphate phosphatase sopB (Salmonella enterica subsp. enterica serovar Typhimurium)... (Created by Alice C.) - wait on this - complex of proteins to be functional.

Target - Protein Phosphatase 2C, putative (T. cruzi).........................................................(Created by Selena Ilbeig) - wait on this. Not enough similarity to PDB to maka homology structure

Target-MtnX phosphatase (Bacillus Anthracis).....................................................................(Created by Brandy C.) - wait. Can't find substrate

Target- Protein Phosphatase 1 (T. brucei) -----------------------------------------------------------------Shivani Bindal - 0.3 Druggability, 80% on 69%

Target - serine-threonine protein phosphatase 2C-(T. gondii)-------(Created by Gautam W.) Researcher: (Was a F13 possibility - but linked to wrong thing so right thing was used)

Target - Putative acid phosphatase (Helicobacter pylori)............(Created by Hyun-Young L.) Researcher: none. Was a F13 possibility

Target- Protein Tyrosine Phosphate (Yersinia Enterocolitica).......... [NOT CLONED in 2012] - low homology 90% on 28% Researcher: none Was a F13 possibility

SUMMER TARGET DISCOVERY REVISITS:

MORNING GROUP

Ashlee, Alyssa, Anita, Young, Katherine V., Manuel, Melissa, Nicolet, Priya, Ramiro , Vicky, William

Vittaforma corneae

Target- EcMetAP2-----------------------------------------Priya P. (double check assay)

Trypanosoma brucei

Target: Dihydrofolate reductase - thymidylate synthase (Trypanosoma brucei) --------------- (created by Melissa Huynh)

Helicobacter pylori

Target - 1-deoxy-D-xylulose-5-phosphate reductoisomerase (H. pylori ) ................Hyun-Young L.

Mycobacterium tuberculosis

Target- Dihydrofolate Reductase M. tuberculosis........................Alyssa K.

Target - Dihyrofolate Reductase (Mycobacterium tuberculosis)............................Ramiro Rodriguez (see Alyssa's page) - join them together

Target - Inositol-1-monophosphatase (Mycobacterium tuberculosis)...................................Nicolet F.

Target: NADH-dependent Enoyl-Acyl carrier protein reductase (M. tuberculosis)..........Katherine V.

Target-protein tyrosine phosphatase (Entamoeba hystolytica).............................................Vicky G.

Target - Serine threonine protein phosphatase (Plasmodium falciparum) .........................................Anita V.

AFTERNOON GROUP

Tony, Brendan, Jiaqi, Karuna, Renee, Kevin, Kavya, Madeline, Mariana, Serena, Oscar, Jason, Laraib, Serena, Grace, Jacky E,

Target- dihydrofolate reductase-thymidylate synthase (Leishmania major)---------------------------Kavya K.

Chlamydia trachomatis

Target - 5-nucleotidase SurE (Chlamydia trachomatis)---------------------------------------------------Tony G.

Target-tyrosine phoshatase B (Mycobacterium tuberculosis).............................................Renee F.

P. falciparum

Target - dihydroorotate dehydrogenase (malaria)----------------------------------------------------------Kevin E.

Shigella dysenteriae

Protein-tyrosine-phosphatase ----------------Jiaqi Zhang.

Leishmania major

Target - Protein Tyrosine Phosphatase Putative (Leishmania major) ----------------------------- Serena Zadoo

Klebsiella pneumonia

Target-New Delhi Metallo Beta Lactamase ....................................Madeline J.

Target - HPr Kinase (Mycobacterium pneumoniae).............................................Brendan C.

PRING 2013 VDSers

Target- NADH-dependent fumaratae reductase (Trypanosome brucei)------Grayson N.

Target- Eonyl-(acyl carrier protein) reductase (Burkholderia Pseudomallei 1719b)------Grant T.

Vibrio cholerae

Target-trypanosoma cruzi, protein tyrosine phosphatase-like protein, putative (t. cruzi)-------Nicole W.

Target-Tyrosine-protein phosphatase (bacillus subtilis)--------------------------------------------------Jessica N.

Target-GDP-mannose pyrophosphorylase(GDP-MP) (Leishmania infantum)..................Keely W.

Target- 3D7, glutathione reductase P. falciparum---------------------------------------------------------------------Seo Kelly K.

Dengue Virus 2

Target-Serine Protease Nonstructural Protein 3(NS3)------------------James T.

Target - Non-structural protein 2B- Serine protease subunit NS2B (Dengue Virus 3) ...........Nancy I.

Salmonella enterica

Enterobacter aerogenes

Phosphatidylglycerophosphatase B--------------------------------Qasim A.

Target-cytosolic malate dehydrogenase (Trypanosoma brucei)----------------------------------------Aditi S.

Target - Inorganic pyrophosphatase (T. brucei)....................................................Shannon R.

Target - serine threonine protein phosphatase 5 (T. brucei) ................ Dax FG

Target - <tyrosine phosphatase> <mycobacterium> ................<Khady D.>Mycobacterium tuberculosis

Target - 1-DEOXY-D-XYLULOSE 5-PHOSPHATE REDUCTOISOMERASE DXR (Mycobacterium Tuberculosis) ................Imran Zafar

Trypanosoma cruzi

Target -Succinate dehydrogenase flavoprotein, putative (T. cruzi) --------------------------------------Daniel D.

Target - protein kinase, putative (T. cruzi) ------------------------------------------------------------------------Alakh R.

Plasmodium vivax

Target: Dihydrofolate Reductase (Plasmodium Falciparum 3D7)-------------------------------------Shawn Okpara

Target: Protein tyrosine phosphatase, putative (Plasmodium Vivax)--------------D'Ondria P.

Target: Leishmania major, aldehyde reductase ------- Spencer Coronado

Target: Leishmania major, trypanothione reductase ------- Aaron H.

Target: Leishmania major strain Friendlin, phosphatase 2c, putative ---------Jesus De La O

Bacillus Anthracis

Salmonella typhimurium

Target - Salmonella enterica subsp. enterica serovar Typhimurium (S.typhimurium) ...................................Karinna L.

Target - 3-oxoacyl- (acyl-carrier-protein) reductase (Mycobacterium tuberculosis)..............Caroline C.

Target -Dihydrodipicolinate Reductase DAPB (DHPR) M. tuberculosis............ So-Youn P.

Target: Thioredoxin reductase (Mycobacterium tuberculosis)---------------------------Ariel C.

Target- Phosphotyrosine Protein Phosphate (Mycobacterium tuberculosis).........Kelsey C.

​Thermoplasma Acidophilum

Target- trehalose-phosphate phosphatase (Thermoplasma Acidophilum).......................Miguel T.

TARGETS THAT WE WILL PASS ON

Target -enoyl-acyl carrier reductase, Toxoplasma gondii------------------------------(created by Grace T.)

Target - UDP-N-acetylenolpyruvylglucosamine reductase (Steptococcus Pneumonia)-----------Brendan C.

Target- succinate dehydrogenase (Plasmodium falciparum)-------------------------------------------Anita V. ----coupled assay (Dr B)

Campylobacter jejuni

Target- 2-Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase)----------------------------Manuel Z. --not enough info

Target - NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (Helicobacter pylori).............William I. (protein complex - 'multifunctional protein')

Summer 2013

no files GDocs: Anita, Kavya, Will, Ramiro, Alyssa, Manuel, Brendan,

Lots: Priya, Vicky, Katherine V., Young, Jacky, Melissa,

Spring 2013

set up 'Teams' - Red, Black, Yellow, etc.

Each time is assocaited with a mentor

-- this is their point of contact for questions

-- they could do peer review within their group

have mentors check their Lab Notebooks before submission.

TestBed:

Fall 2012

PyMol Image Contest

Schedule Fall12

Timeline F12

Timeline Vets F12 (deprecated added stuff to Timeline and split the view in half)

Daily Schedule Summ12

Planning

Guest speakers?

• 1. Profs from ACCbiotech (careers in research),

  2. Karishma Kaushik

  3. Devinder

  4. Grace

  5. Lawrence

Cloning vs. expression

-- clone new targets (phosphatases only)

--

PyMol Refresher

VIrtual SCreening refresher

ICM tutorial

Optional: VINA tutorial - before students can run VINA - have them write one bash script

ToDo List (Dr. B):

• • Homology model

  • Enzyme Assay kinetics lab (phosphatase)

  • Journal Clubs

  • revise Tranformation for Midiprep protocol to require 160 ml of LB for pNIC-bsa4growth

    • • or just say to use all 4 80 ml tubes in Midiprep preparation

Collaboration project with Supramolecular Sensors (Anslyn/Ghanem) stream

-- Eman will run it by Anslyn

• 1. expression of the enzyme to give to Supramolecular stream to do phage display binding and sequencing

     1. • phage display screening only selects binders but gives you no information about binding strength or location of binding (active site vs. allosteric)

  2. virtual screening of top cyclic peptides from phage display screening into an enzyme active site

     1. • benefit of virtual is that we can limit the number of peptides that will need to be synthesized

  3. enzyme assays with the best cyclic peptides chosen from

Issues:

• • what enzyme to use - needs to have large active site to accept a peptide. Maybe DXR (cheaper reagents - big NADH site)

    • • however if you target the NADH site - you would have more sideeffects in the patient.

  • how to create 'virtual' peptides - probably on ICM

Summer 2012

Virtual Work to be done: (post result to Wiki)

ICM

• • regularization vs Optimization vs Minimization (Cartesian, local, global) - do RMSD differences?

  • Flexible Receptor Docking with 1U72

  • Ensemble docking with various PDB models of 1U72

  • Homology model protocol

Avidin/Biotin Test - downloaded some files to a DDFE folder

• • GOLD

  • ICM

  • VINA

ICM vs. GOLD vs. VINA

doc 20ligs vs. DHFR receptor - compare ranking, post on Wiki

• • GOLD

  • ICM

  • VINA

Get Markush library to work in Maestro (since no license in ICM)

Maybe have Christina work on RNA screening validation - see PseudoKnot project page

**add in molecular dynamics (using Maestro) of ICM and GOLD docked compounds to further refine selections

VINA running on TACC - see 'TACC VINA Work' page - use Alex and Christina

*

2012 Targets

Targets that were not successfully cloned in 2012

Paul, Ling, Rishi [NOT SUCCESSFULLY CLONED]

Ivy, Stephanie, Jennifer [NOT SUCCESSFULLY CLONED]

Divya, Tom, Sajan [NOT SUCCESSFULLY CLONED]

Suman, Brandon, Ruifei [NOT SUCCESSFULLY CLONED]

Akhilesh, Mihir, Shane [NOT SUCCESSFULLY CLONED]

Leishmania

Wolbachia endosymbiont of Brugia malayi (see Wolbachia Targets page too)

Target - 1-deoxy-D-xylulose 5-phosphate reductoisomerase [DXR] (Wolbachia) ------------------------ Urvashi

Target - FabI enoyl-acyl-carrier-protein reductase NADH (Wolbachia) ------------------------------ Daniel

Yersinia

Target - Fab V (Yersinia Pestis) --------------------------------------------------------------------------------- Max, Michael

Trypanosoma cruzi

Target - dihydrofolate reductase-thymidylate synthase (T. cruzi)--------------------------------------Alex, Ling,

Target - Glyceraldehyde-3-phosphate dehydrogenase (T. cruzi)---------------------------------------Andrew, Rishi

Francisella tularensis

Target -1-deoxy-D-xylulose 5-phosphate reductoisomerase (Francisella Tularensis)----------Janice, Aldo [NOT SUCCESSFULLY CLONED]

Trypanosoma brucei

Target - Glyceraldehyde-3-phosphate dehydrogenase (T. Brucei) ....................................Larry

Plasmodium falciparum

Target - 1-deoxy-D-xylulose-5-phosphate reductoisomerase (P. falciparum)---------------------Kaarthik, Andrew, Alex

Staphylococcus agalactiae - Neonatal Septicemia

Listeria monocytogenes

Shigellosis dysenteriae

Target - B-OCTYLGLUCOSIDE (Shigellosis) ----------Jiaqi Zhang

Candida albicans

Target - Methionine Synthase --------------------------- Priya A

2011 Targets

Target-Histidine Phosphatase (Francisella Tularensis) ---------------------------------------------------Christina, Joey,

Target-MPTPB(Mycobacterium Tuberculosis) H37Rv----------------------------------------------------- Joshua

Target - Protein-Tyrosine Phosphatase Yersinia pestis YopH (Bubonic Plague)-------------------- J.C.

Target-Mycobacterium Tuberculosis PSTP ------------------------------------------------------------------Sadhana, Michael

Target - Carbonic Anhydrase IX (Homo sapiens) -----------------------------------------------------------Vanna, Keyur

Target- Carbonic Anhydrase YadF and cynT(E. coli)

Target - PTP1b (Homo sapiens) ---------------------------------------------------------------------------------VDS Staff

Targets not pursued:

Francisella tularensis

deprecated - Target -3-oxoacyl-acyl-carrier-protein reductase (Francisella Tularensis)---------------------------Jennifer R.

Leishmania

Target-dUTP diphosphatase (Leishmania major)------------------------------------------------------------- Shane -Requires coupled assay -- Dr. B

Clostridium Perfrigens

Target- phospholipase C (C. Perfrigens)---------------------------- Sajan this protein is a toxin

Staphylococcus aureus

Target- YopH (S. aureus)---------------------------------------------------------Stephanie B ------primer design is off, see if you can fix it. Redo - DNA works. --Dr. B

Target- Inositol-phosphate phosphatase (S. aureus).........................Brandon N. - essentiality?? -- Dr. B

Target - NMN phosphatase, Class B acid phosphatase precursor (Staphylococcus aureus)------------------Mihir - not uploaded to primers -- Dr.B

Target - fructose-1,6-bisphosphate aldolase (P. falciparum) ....................................Ruifei - Requires coupled assay -- Dr. B

Target - V-type H translocating pyrophosphatase ---------------------Tom S. - not uploaded to primers - Dr. B

• • • • New Item - each target page should include an image of the TMpred graph (http://www.ch.embnet.org/software/TMPRED_form.html). In put your amino acid sequence to it.

•Wolbachia .....................................................................(Urvashi, Divya, Daniel, Ramya)

Wolbachia Targets

https://www.wolbachiawebsite.org/

http://www.a-wol.net/

Q5GRQ1 - Citrate Synthase (Wolbachia) - essentiality?

Q5GSP1 2-oxoglutarate dehydrogenase complex (Wolbachia) - need to check enzyme assay conditions.- Dr. B

Q5GSI8 Succinate dehydrogenase flavoprotein subunit, SdhA (Wolbachia) - need to check enzyme assay conditions.- Dr. B

Q5GTF8 NADH-quinone oxidoreductase subunit D (Wolbachia)

Q5GS48 Acetyl-CoA carboxylase, carboxyltransferase component (Wolbachia)

Q5GS70 Thioredoxin reductase (Wolbachia) - Good druggability in P. falciparum - Dr. B

Q5GSR3 Delta-aminolevulinic acid dehydratase (ALAD) (Wolbachia) - hazardous substrate in assay - Dr. B

Transketolase - Druggable, coupled assay though (substrate availability?)

Q5GTA4 1-deoxy-D-xylulose 5-phosphate reductoisomerase - Dr. B

FabG 3-oxoacyl-[acyl-carrier-protein] reductase EC 1.1.1.100 - called 'Short-chain alcohol dehydrogenase family enzyme'

FabI enoyl-[acyl-carrier-protein] reductase NADH EC 1.3.1.9

deprecated - Target - 2-oxoglutarate dehydrogenase complex (Wolbachia) --------------------------------------- Daniel

deprecated - Target- NADH-quinone oxidoreductase subunit D (Wolbachia)----------------------------------------Divya

deprecated - Target - Acetyl-CoA carboxylase, carboxyltransferase component wolbachia--------------------------------- Daniel

deprecated - Target - Transketolase (Wolbachia) not used-----------------------------------------------------------------------------Urvashi

deprecated - Target - FabG 3-oxoacyl-acyl-carrier-protein reductase (Wolbachia) not used------------------------------Urvashi

deprecated - Target - Citrate Synthase (Wolbachia) not used ----------------------------------------------------------------- Ramya

deprecated - Target - Thioredoxin reductase (Wolbachia) not used------------------------------------------------------------------Urvashi

•Chagas (T.cruzi) ......................................................................... (Andrew, Ling, Rishi, Alex)

--------- Do TDR targets search with CRITERIA: phosphatase, Essentiality, Druggability > 0.5. Then manually search for those with good Essentiality, Good match to PDB (>50%), and substrates available (Sigma). -- Dr. B

1-deoxy-D-xylulose 5-phosphate reductoisomerase - none found

DHFR?

FabI enoyl-[acyl-carrier-protein] reductase NADH EC 1.3.1.9

FabG 3-oxoacyl-[acyl-carrier-protein] reductase EC 1.1.1.100

3-oxoacyl-(acyl-carrier protein) reductase, putative - TDRTargets

•Francisella tularensis ......................................................(Aldo, Janice, Jennifer R.)

------------------NAD pathway of Francisella tularensis is unique http://www.bio.utsa.edu/labpages/klose/xzogaj.htm

http://www.sciencedirect.com/science/article/pii/S0304416501001131

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223557/?tool=pubmed

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.1.13

1-deoxy-D-xylulose 5-phosphate reductoisomerase - yes - Dr. B

FabG? 3-oxoacyl-[acyl-carrier-protein] reductase EC 1.1.1.100

•Yersinia pestis..................................................................(Michael, Max)

FabG? 3-oxoacyl-[acyl-carrier-protein] reductase EC 1.1.1.100

FabI enoyl-[acyl-carrier-protein] reductase NADH EC 1.3.1.9

•Leishmania .....................................................................(Sam, Mihir, Shane )

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.6.1.23

http://modbase.compbio.ucsf.edu/modbase-cgi/model_details.cgi?queryfile=1338502903_581&searchmode=default&displaymode=moddetail&referer=yes&snpflag=&

Try this for Leishmania: LmjF25.1320 - Dr. B

1-deoxy-D-xylulose 5-phosphate reductoisomerase - none found

Kaarthik - Fen1 P. falciparum

P. falciparum (and others)

1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP reductoisomerase or IspC)

EC 1.1.1.267, essential = YES, assayable = YES

Gene ID: 812223

__https://www.ncbi.nlm.nih.gov/gene/812223__

DNA Output File

Past target 2011 - Target- Carbonic Anhydrase (Salmonella) ------------------------------------------------------------------Krishna

Past target 2011 - Target - Serine-Threonine Phosophatase 2C From Toxoplasma Gondii------------------------------Larry

Past target 2011 - Target-Apyrase Phosphotase (Shigella flexneri) ------------------------------------------------------------

deprecated - Target-PARP1 (Homo sapiens)

Targets for pNIC-Bsa4 cloning practice

Larry Potential Targets Summer 2012

I used TDR to find some potential targets. (It was the easiest to find essentiality data with)

How does the amino acid sequence BLAST against the PDB? - are there any similar enzymes which have a crystal structure?

Be sure to check for any Transmembrane regions (not sure if that is on the Target Discovery Protocol or not)

http://www.tdrtargets.org/targets/view?gene_id=32911

Unfortunately this one looks to have poor essentiality data.

Good - has druggability data - unlike the rest of the TDR ones listed

For this one - try to follow the paper link in Assayability and figure out exactly how the assay is done (spectrophotometric?, substrates, etc)

They do it "spectrophotometrically taking advantage of the release of protons involved in the reaction", Here. I don't understand how the Michaelis–Menten equation is used, or why there are protons released.

--- It is splitting water and putting one proton into solution per reaction. The M-M equations is just for the kinetics. We will get to that later.

Here is a link tot he BioMol Green reagent we can use phosphate detection - but can it detect pyrophosphate (2 phosphates?)

On the product page it says one known

application

is "tyrosyl-tRNA synthetase (coupled with pyrophosphatase)" so I'm guessing it can detect pyrophosphatases.

It means that they use a coupled assay with a phosphatase to cleave the pyrophosphate into two phosphates so that biomol green can be used.

-- If so, then we may have the choice of assay - either the colorimetric pH indicators or this Biomol reagent. --Dr. B

http://www.tdrtargets.org/targets/view?gene_id=257047

HisE - Looks interesting - how about the assay?

It is a Phosphoribosyl-ATP pyrophosphatase, so we can use a pyrophosphate assay, which should be readily available.

Is the substrate ?

Not sure about this one, couldn't find it in Sigma.

It is a small enzyme too 10kDa - so need to be careful with centrifugal filter MWCO.

http://www.tdrtargets.org/targets/view?gene_id=255685

looks good too

For this one can we just use dUTP as the substrate?

http://www.tdrtargets.org/targets/view?gene_id=256061

Essentiality is not great -exclude this one

http://www.tdrtargets.org/targets/view?gene_id=6216

Good too: Same His E as 257047 but for M. tuberculosis instead of M. Leprae - what is the difference between the two strains?

M. Leprae . It's very difficult to culture since it lacks many vital genes. M. tuberculosis is also highly aerobic, so it infects the respiratory system while M. Leprae does not need as much oxygen, although is still aerobic. They both have the waxy coating that all mycobacteria have.

http://www.tdrtargets.org/targets/view?gene_id=5895

Some I had: What does "probable" mean in the title for the 3rd compound protein? How should I interpret the essentiality data? Some of the essentiality studies are split on essential or not. Does the PDB file need to have a substrate?

-- probable' means they haven't proven its role but based on similarity to other sequences, they think they know what it does.

Essentiality data is hit or miss - but just go for ones that at least have some essentiality verified

PDB file does not 'need' a substrate - as long as you know where the active site is.

- Dr. B

http://www.rcsb.org/pdb/explore/explore.do?job=summary&pdbId=2i34

Here's one from Pubmed for anthrax, but there's no article on it, and I'm not sure how to figure out essentiality.

--- Would be easy assay, just not sure if it is an important enzyme. --Dr. B

---- Look in the PATRIC database and try to find this. There may be some essentiality data there. Also, search pubmed for more papers on this enzyme to see if there is any essentiality data. - Dr. B

When I search for anthrax in Brenda, a lot of the times it will have the enzyme, but when I click on the link I can't find bacillus anthracis on the list.

--- That is ok - it may not be specifically in there - as long as there are other bacterial versions in there then we are relatively confident. --Dr. B

Here are some for F. tularensis:

http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.2&r=666833

It seems that this protein already has known inhibitors

-- Also, really similar to Joey and Christina's target -- Dr. B

http://www.rcsb.org/pdb/explore/explore.do?job=summary&pdbId=3nea

No essentiality data I don't think.

-- Assay not as simple --Dr. B

Trypanasoma cruzi

http://www.tdrtargets.org/targets/view?gene_id=54598

Does the known druggable targets suggest that it is too similar to humans?

-- Coupled assay -- Dr. B

http://www.tdrtargets.org/targets/view?gene_id=11394

http://www.pdb.org/pdb/explore/explore.do?structureId=2W7T

http://www.jbc.org/content/282/16/11858.long

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.4.2

CTP Synthetase for t. brucei

http://www.tdrtargets.org/survey/view/13

/?tab=mo&authuser=0#folders/0B4O2KqKh2q_-U2VjeTQwRzU1cjA

Daily+Schedule+Summ12

14

24

S1368764600901778

glyceralde3phosdehydrbaci.pdf

DisplayMSDSPage.do

result_flat.php4

lit.php4

lit.php4

7932587

abstract

jm9802620

SUBSTRATES INFO:

See Wu and Knight paper in the Purchasing/Sigma folder for ino on NADH and NADPH degradation:

Conclusions: store NADPH above pH 7.5. Can use Tris + NaOH to do this.

NADH - sigma N4505

Product Information Sheet:

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n4505pis.Par.0001.File.tmp/n4505pis.pdf

Preparation Instructions

This product is soluble in 0.01 M NaOH (100 mg/ml).

Water alone should not be used to prepare solutions

since it tends to be acidic and would decompose

b-NADH. If solutions must be stored for any length of

time, phosphate buffers should be avoided since they

accelerate the destruction of b-NADH.6,7 TrizmaÒ

(0.01 M, pH 8.5) and MES buffers are better options.

Since b-NADH solutions are susceptible to oxidation

even at low temperatures, solutions should be prepared

at concentrations no greater than 5 mM, at a pH of

9–11, and stored at 4 °C.6 The presence of light and

heavy metals can accelerate the oxidation process.1 If a

low temperature freezer is available (temperatures at

–40 °C or colder), more concentrated solutions can be

prepared and stored for years without any loss of

activity.6

Storage/Stability

b-NADH should be stored ,desiccated and protected

from light, at –20 °C.1

Solutions should be freshly prepared and used promptly

unless extreme care is taken.

B-nad (nicotinamide adenine dinculeotide) B-NAD+

N7004

http://www.sigmaaldrich.com/catalog/product/sial/n7004?lang=en®ion=US

Storage/Stabilityβ-NAD is very hygroscopic and should be stored desiccated.Aqueous solutions between pH 2-6, stored as single - use aliquots at - 70°C, are stable for at least 6 months. Neutral or slightly acidic solutionsare stable at 0°C for at least 2 weeks. Solutions arerapidly degraded upon heating and are very labile inalkaline solutions, especially in the presence ofphosphate, maleate, or carbonate. The rates ofdegradation of solutions at different pH and temperature conditions have been reported. Solutionsare also sensitive to light.

Also see Biotek comparison of NADH vs NADPH for making stocks using Tris, EDTA ph 8

http://www.biotek.com/resources/docs/NADH_App_Note.pdf

NADPH - sigma N6505

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf

Preparation Instructions

b-NADPH is soluble in 0.01 M sodium hydroxide

(50 mg/ml), yielding a clear, light yellow solution.

Storage/Stability

It is recommended to store Products N1630, N7505,

and N6505 desiccated at –20 °C protected from light.

Product N9910 can be stored at room temperature.

The normal impurities and/or decomposition products

are b-NADP and monophosphoadenosine 5¢-

diphosphoribose.

It is recommended to prepare solutions fresh and use

promptly, unless you are sure this is an unnecessary

precaution for your work. However, it has been

reported that a 0.5 mM solution in 0.02 M NaOH

(pH 12.3) showed no loss of purity in a week at 4 °C or

-85 °C, but a 13% loss at –20 °C.3

For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.

Enzo Life Sciences:

NADPH is unstable in acid media. Stock solutions should be at pH 8.

Soluble in water (50mg/ml) or 0.01M sodium hydroxide.

NADP+

b-NICOTINAMIDE ADENINE DINUCLEOTIDE

PHOSPHATE

Sodium Salt

Product Number N0505

http://www.sigmaaldrich.com/catalog/product/sigma/n0505?lang=en®ion=US

Preparation Instructionsβ-NADP+ is soluble in water at 50mg/ml.It is also soluble in methanol, much less soluble in ethanol andpractically insoluble in ether and ethyl acetate.Aqueous solutions stored as frozen aliquots are stable for at least one year.Repeated freeze thaw cycles are not recommended.Storage/StabilityStore at–20ºC

Enzo Life Sciecnefor NADP+

http://www.enzolifesciences.com/ALX-480-003/nadp-.-disodium-salt/

soluble in water to 50mg/ml

sesnsitive to alkaline - so store in water that is a little acidic.

6-phosphogluconate

P7877

http://www.sigmaaldrich.com/catalog/product/sigma/p7877?lang=en®ion=US

?? - no info on storage

Crotonyl coenzyme A trilithium salt

Simga: 28007 $246

http://www.sigmaaldrich.com/catalog/product/sigma/28007?lang=en®ion=US

H2O: soluble50 mg/mL, clear, colorless

storage life and conditions: ?

Sigma Tech Support suggests aliquoting and storing in -80degC

Kate K also had C6146 - 5mg 2 Butenoyl CoA Lithium Salt in her freezer. Is this for Fab I? $232.50

Yes, this is the same thing. For some reason this product number is cheaper

1-Deoxy-D-xylulose-5-phosphate sodium salt (DXP)

Sigma 13368

solubility: 50 mg/ml in H2O

storage life and conditions: ?

Sigma Tech Support suggests aliquoting and storing in -80degC

FagG

acetoacetyl-CoA + NAPDH --> 3-hydroxybutyryl-CoA + NADP+

acetoacetyl-CoA A1625-5MG - $125

NAPDH N6505-25mg - $87

May be try the reverse reaction:

NADP+ Product Number N0505 $79.70 for 100 MG - CHEAP

DL-β-Hydroxybutyryl coenzyme A lithium salt H0261-10MG $218 - Cheaper thatn AAC

TARGETS

Yersinia pestis study - old plague not different from new plague..

http://www.ncbi.nlm.nih.gov/pubmed/21993626

Abstract

Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard. This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348-1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347-1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y. pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y. pestis infections.

Genes of Interest from Wolbachia

Q5GRQ1 - Citrate Synthase

Assay on Sigma - DTNB

http://www.sigmaaldrich.com/catalog/product/sigma/cs0720?lang=en&region=US

D8130 - DTNB Sigma cheap

B6935 - Assay Buffer - unknown

O4126 - Oxaloacetic acid, cheap

Q5GSP1 2-oxoglutarate dehydrogenase complex

would need to make all enzymes in complex?

NADH based assay

S1129 Sigma - Succinly coA

K3752 Sigma - alpha-Ketoglutaric acid

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.2.4.2

Similar PDB:

http://www.rcsb.org/pdb/explore/explore.do?structureId=2JGD

Thiamine dependent? AMDP dependent?

Inhibited by Oxaloacetate (in paper fromPDB)

Q5GSI8 Succinate dehydrogenase flavoprotein subunit, SdhA

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.5.1

W327700 - Sigma Succinate

Assay not good?

http://jb.oxfordjournals.org/content/145/2/229.long (from Brenda link)

uses colorimetric at 600 nm for DCIP

2,4-Dichlorophenolindophenol - was used

2,6-Dichlorophenolindophenol - is available - at Sigma (http://www.sigmaaldrich.com/catalog/product/fluka/33125?lang=en&region=US)

Similar PDB (not the one from BLASTP) - just form text search for SDHA

http://www.rcsb.org/pdb/explore/explore.do?structureId=1NEK

Q5GTF8 NADH-quinone oxidoreductase subunit D

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.6.99.5

Assay - 2,6 DCIP similar to Succinate dehyrdogenase

PDB Blast

http://www.rcsb.org/pdb/explore/explore.do?structureId=2FUG

Q5GS48 Acetyl-CoA carboxylase, carboxyltransferase component

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=6.4.1.3

http://www.rcsb.org/pdb/explore/explore.do?structureId=3N6R

A2056 Sigma Acetyl CoA

Free phosphate detection after ATP

Q5GS70 Thioredoxin reductase

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.8.1.9

http://www.rcsb.org/pdb/explore/explore.do?structureId=1TDE

T0910 Sigma - Thioredoxin $169.50 /mg

NADPH assay

Q5GSR3 Delta-aminolevulinic acid dehydratase (ALAD)

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.24

A3785 Sigma- cheap

http://www.jbc.org/content/277/22/19792.long

http://pubs.acs.org/doi/full/10.1021/bi010656k

at A555nm

assay 2 step with Ehrlich's reagent and Mercury (!)

END Genes of Interest from Wolbachia

Other Genes of Interest

Candida albicans

Candida albicans are the most common fungi associated with biofilm-related infections, including urinary tract infections, catheter infections, child middle-ear infections, dental plaque, and endocarditis. Although resistance genes, such as MDR, CDR1, and CDR2, have been identified, research has shown that deletion of these genes does not confer sensitivity to drug therapy in mature biofilms.

--Tobudi, et al. Mycoses. 2012; 55:199-204

Dr. B

Trypanosoma brucei - African sleeping sickness

"Treatment requires two drugs, suramin and melarsoprol, that are both very toxic and so rare that they must be directly requested from the World Health Organization"

http://www.wired.com/wiredscience/2012/04/exotic-travel-serious/#more-107000

Other potential targets:

NAD pathway of Francisella tularensis is unique

http://www.bio.utsa.edu/labpages/klose/xzogaj.htm

The number of people who are dying from illnesses that involve vomiting and diarrhea more than doubled between 1999 and 2007, and most of the increase was due to Clostridium difficile. -Wired

Plasmoidum falciparans (malaria)

- structure available now 3AU8

Target - 1-deoxy-D-xylulose-5-phosphate reductoisomerase (good target for many)

Leishmania major

Target - serine threonine protein phosphatase //Leishmania major//

YwqE- bacterial phosphatase

different from most phosphatases (Bacillus cereus or Bacillus anthrax (?)) - not sure of essentiality

species?

essentiality?

unique from other phosphatases

3Qy6 PDB

LipA - phosphatase

essentiality? - but for virulence

Listeria monocytogenes

LipA - phosphatase involved in Virulence of Listeria monocytogenes - not sure of essentiality but involved in virulence

ribose 5-phosphate epimerase (RipA or RipB?)

Plasmoidum falciparans (malaria)

essentiality?

Ribose5P assay at 280nm

cheap substrate

DdlA - D-Ala:D-Ala ligase

bacteria or protozoan.

Virtual done on DdlB (Kovac paper) but now there are structures of DdlA on PDB.

Assay is Malachite green (Biomol), Sigma has substrates

Chagas disease (Trypanasoma cruzi)

inositol-phosphate phosphatase (maybe not good, the Druggable links take you to an alternative for Lithium treaments for manic depression - so may not be a good Essentiality in infectious organisms)

T. cruzi (and others)

essentiality?

use malachite green assay and/or pNPP

D-myo-Inositol-1-phosphate (sodium salt) form Cayman Chemical #10007777 $53 for 1 mg

SOD (Superoxide dismutase) in bacteria is used to prevent oxidative stress induced upon it by the human host immune system. (H. pylori has PDB, M. tuberculosis has PDB). Also, perhaps //Burkholderia// homology model could be made. Coupled assays, though...

- Homo sapiens Carbonic Anhydrase 2

White Nose Bat Disease - Fungal

http://vdsstream.wikispaces.com/page/edit/White+nose+bat+disease

Previous Stuff:

E. coli carbonic anhydrase - (can or YadF) have sequence, assay ok, expression?, essentiality?

http://ecogene.org/geneinfo.php?eg_id=EG12319

forms Synthetic lethal with cynT....

E. coli carbonic anhydrase - cynT

http://ecogene.org/geneinfo.php?eg_id=EG10176

Human carbonic anhydrase 9 (cancer metastasis?) - different enough from others - there is one homology model and one true structure on PDB

M. tuberculosis - carbonic anhydrase Rv1284 - essentiality

M. tuberculosis - carbonic anhydrase Rn3588c canB - (yes and no essentiality)

Leishmania, Plasmodium, or Toxoplasmosa

DHFR, or DHFR-TS

FabI - enoyl-acyl carrier protein reductase

FabG - 3-oxoacyl-[acyl-carrier-protein] reductase

Clostridium difficile toxin - would need protease assay or maybe DSF for cleavage products?

http://www.pdb.org/pdb/explore/explore.do?structureId=3PA8

http://www.ncbi.nlm.nih.gov/pubmed/21095570

Plasmodium falciparans - Malaria

ubiquitin carboxyl-terminal hydrolase, Ub-AMC fluorescence assay (essentiality?)

FTIs can also be used to inhibit farnesylation in parasites such as trypansoma brucii and malaria.

Parasites seem to be more vulnerable to inhibition of Farnesyl transferase than humans are.

In some cases this may be because they lack Geranylgeranyltransferase I.

Thus it may be possible for the development of antiparastic drugs to 'piggyback' on the development of FTIs for cancer research.

TDR Targets search for any Essentiality and Druggability >= 0.5 - yields 1144 hits

TDR Targets search for any Essentiality and Druggability >= 0.8 - yields 324 hits

TDR Targets search for any Essentiality and Druggability >= 0.9 - yields 41 hits

TDR Targets search for any Essentiality and Druggability >= 1.0 - yields 41 hits (same)

TDR Targets search for 'carbonic' and any Essentiality - yields 6 hits (in tuberculosis, leishmania, etc.)

TDR Targets search for 'phosphatase' and any Essentiality - yields 170 hits

TDR Targets search for 'phosphatase' and any Essentiality and Druggability >= 0.5 - yields 21 hits

pstP

Rv0018c

Identified in the membrane fraction of M. tuberculosis H37Rv

PHOSPHOSERINE/THREONINE PHOSPHATASE PSTP

non essential gene by Himar1-based transposon mutagenesis in H37Rv strain

Druggability: 0.2

transmembrane portion though - possible to make truncated version?

T. cruzi (and others)

inositol-phosphate phosphatase

EC 3.1.3.25

PNPP (high Km)

or

Use the following with Malachite Green detection of free phosphates

D-myo-Inositol-1-phosphate (sodium salt) form Cayman Chemical #10007777 $53 for 1 mg

Pubchem:

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=5288642

TDR Targets:

inosine 5'-monophosphate dehydrogenase IMP dehydrogenase

substrate: NADPH and

Xanthosine 5′-monophosphate (X1000 discontinued from Sigma)

1 of 5 for essentiality references

T. cruzi - ecto-nucleoside triphosphate diphosphohydrolase (infectivity and virulence)

Furin - human protesase used by Dengue and Anthrax for infectivity. Assay? Bioluminescence dependent on Furin -

expensive assay? Side effects? Substrates:

with CPA95 as substrate/with N-tert-butoxycarbonyl-Arg-Val-Arg-Arg-methylcoumarin amide as substrate

with Ac-Arg-Val-Arg-Arg-4-nitroanilide as substrate

Naegleria fowleri - water born amoeba kills in 3-7 days eating brain

08/17/2016

Staphylopine as a drug target for bacteria (specifically S. aureus, Y. pestis, and P aeruginosa

- one would target the machinery (proteins) that make this small molecule metallophore (e.g. it binds up metals and helps them get transported into the cell so it can survive)

There are 3 enzymes supposedly which make this from amino acid lysine.

There are also some proteing involved in trafficking staphylopine (move it in and out of cell membrane)

- see C&EN news May 30, 2016 edition page 9.

- see Pascal Arnoux Bioscience & Biotechnology Institute in Aix-Marseille, France

- see Eric Skaar lab of Vanderbilt University.