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:

Walter Fast
- Oscar, Brianna, Madeline,

Rhana Gosh
- Fin, Alberto Tueme Cruz

Hubreigtse
- Avery Wolfe

Finkelstein
- Brianna (Xenia) G.

Emily Que:
we are making a bunch of probes to bind metalloenzymes for imaging, proteomics, and ‘chemical biology” type applications and would maybe have a place for someone with experience in virtual design of molecules to bind to proteins.

Yan Jessie Zhang
- Karan K.

Keatinge-Clay
- Luis V.

Lauren Webb:
- Ruifei Wang


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)
    • 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
    • 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
    • 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



•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
HsCD00041026

CA7
HsCD00002513

PTP1b
Zhang

Scp1
HsCD00040190
ASU PSI identifier
CA2

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 external image arrow-10x10.png 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 external image arrow-10x10.png?
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 external image arrow-10x10.png. 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 external image arrow-10x10.png 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

A sampling of rare diseases

http://the-scientist.com/2011/08/01/make-mine-rare/

Company
Indication
Phase
PTC Therapeutics
South Plainfield, NJ
Cystic fibrosis
Duchenne muscular dystrophy
Hemophilia
III
III
II
Amicus Therapeutics
Cranbury, NJ
Fabry disease
Pompe disease
Gaucher disease
III
I
II
Hyperion Therapeutics
South San Francisco, CA
Urea cycle disorders
Hepatic encephalopathy
III
II
Alexion Pharmaceuticals
Cheshire, CT
Paroxysmal nocturnal hemoglobinuria
Atypical hemolytic uremic syndrome
Cold agglutinin disease
Approved
II
II
BioMarin Pharmaceutical
Novato, CA
Lysosomal storage disorders
Phenylketonuria
Lambert-Eaton myasthenic syndrome
2 products approved
Approved
Approved in EU



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.