Source: TEXAS A&M UNIVERSITY submitted to NRP
APPLYING STRUCTURAL GENOMICS AND DRUG DISCOVERY TO UNDERSTANDING DRUG RESISTANCE
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
1003572
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 8, 2014
Project End Date
Jul 8, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biochemistry & Biophysics
Non Technical Summary
Drug resistance is rapidly becoming the most significant health problem worldwide. This applies not only to drug resistantinfectious agents in developing countries. It is estimated that most cancer death are now caused by resistant cancer cells, anddrug resistant bacterial disease resulted in 23,000 deaths in the US in 2013 (CDC).The mortality from tuberculosis (TB) worldwide continues to increase. Even 50 years after discovering the drugs capable ofcuring TB, the global burden of this ancient disease is still substantial. In 2012, an estimated 8.6 million people became ill withTB and 1.3 million people died, according to the World Health Organization (WHO). Each year, roughly 500,000 childrendevelop TB and as many as 74,000 die from the disease. For HIV-infected individuals, co-infection with TB represents theleading cause of death. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB are recognized as a growing problem, threatening overall TBcare efforts. The past 20 years have seen the worldwide appearance of multidrug-resistant (MDR) tuberculosis, followed byextensively drug-resistant (XDR) tuberculosis, which was first described in 2006. About 3.6% of new tuberculosis (TB) patientsin the world have Multidrug-Resistant strains (MDR-TB). Levels are much higher about 20% in those previously treated for TB.The frequency of MDR-TB varies substantially between countries. About 10% of MDR-TB cases are also resistant to the twomost important second-line drug classes also known as Extensively Drug-Resistant TB (XDR-TB). By September 2013, 92countries had reported at least one XDR-TB case. The year 2007 saw the occurrence of strains that are resistant to allantituberculosis drugs, now known as Totally Drug-Resistant (TDR) TB. MDR tuberculosis is caused by Mycobacteriumtuberculosis that is resistant at least to isoniazid and rifampicin, and XDR tuberculosis by mycobacteria resistant to rifampicinand isoniazid, any fluoroquinolone, and one of the three injectable drugs, capreomycin, kanamycin, and amikacin. Drugresistance severely threatens tuberculosis control, since it raises the possibility of a return to an era in which drugs are no longereffective. To achieve global control of this epidemic, there is an urgent need for new TB drugs, which can:(1) shorten treatmentduration;(2) target MDR or XDR strains; (3) simplify treatment by reducing the daily pill burden; (4) lower dosing frequency (forexample, a once-weekly regimen); and (5) be co-administered with HIV medicationsPioneering studies have demonstrated that establishment of resistance requires a reversible phenotypic shift without which thebacilli rapidly lose viability. An essential component of this shift is the change from aerobic to anaerobic metabolism. We haveshown that a key player in this metabolic shift is the glyoxylate shunt, as mutants defective for isocitrate lyase are unable topersist in the murine infection model. While the major focus of the project will be on TB the lessons learned and the novel strategies will have broader implications.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
72340101000100%
Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
Our objectives are:1. To determine the targets of whole cell active hits from phenotype Mtb high throughput screens.2. To perform structural studies of novel Mtb targets for medicinal chemistry and drug discovery.3. Use Mtb phage tail fibers as targeted drug delivery for Mtb.
Project Methods
a. To determine the targets of whole cell active hits from phenotype Mtb high throughput screensWe have used whole-genome sequencing (WGS) of isogenic mutants selected for resistance to various HTS compounds toidentify new targets. In M. tuberculosis, we have identified several new enzymes as targets of whole-cell active inhibitors. Forexample, we selected resistant mutants for one compound and performed WGS and found that the only common mutationidentified was in FadD31 (fatty-acyl AMP ligase). Similarly, we have used sequencing of resistant mutants to identify Pks13(polyketide synthase, synthesis of mycolic acids) and AspS (Asp-tRNA synthetase) as targets of other HTS compounds. Weselected resistant mutants for another compound and, via WGS, we identified mutations in DprE1 (decaprenylphosphoryl-β-Dribose2'-epimerase), which catalyzes a key step in synthesis of arabinogalactan. This gene was recently shown to be the targetof benzothiazinones. Our inhibitor has a different chemical structure, but selects for the same mutation, C387S, in the activesite.PepQ is another novel target we identified in Mtb. Through work with a collaborator, we performed WGS on Mtb that had beenpassaged through mice treated with TMC-207 (bedaquiline) and/or clofazimine. We found that these cells had acquiredmutations in vivo in PepQ (including loss-of-function mutations) that resulted in co-resistance to both drugs. The target of TMC-207 has been shown to be the F0F1 ATP synthase, and the target of clofazimine is unknown. PepQ is a prolidase(aminopeptidase with specificity for cleavage of terminal prolines). We are anxious to work out the resistance mechanism.Finally, we recently identified inhibitors that select for mutations in the ESX-3 type VII secretion system (T7S) by sequencingmutants resistant to several HTS compounds. ESX-3 is a large membrane-associated complex of at least 5 proteins, EccA3 -EccE3. There are five T7Ss in the Mtb genome, but this is the only one essential in vitro (based on transposon mutagenesis).ESX-3 has been demonstrated to be implicated in iron acquisition, though its precise function has not yet been elucidated.Several subunits are integral membrane proteins (EccB3, EccD3); others are ATPases (EccA3, EccC3); but all components inthe complex should be viable drug targets. We observed mutations in EccA3, EccB3, EccC3, and EccD3 (to different inhibitors).We will use medicinal chemistry and structural-based studies to develop leads that target M. tuberculosis Pks13 and AccD6.Both enzymes are important components in the synthesis of the mycobacterial cell wall. For Pks13, the initial hit, a benzofuran,was discovered through a whole-cell screen for molecules with growth inhibition activity against Mtb H37Rv. For AccD6, thestarting point consisted of herbicides known to be active against the plant homolog of the enzyme, and quizalofop was found tohave activity both against Mtb AccD6 and against cell cultures of M. tuberculosis. Both compound series have drug-likeproperties and have potential to be developed into antitubercular drug candidates. To date, we have solved crystal structures ofthe enzymes in apo form, developed enzyme assays, identified inhibitors, and obtained crystal structures of complexes withwhole cell actives. We have chemically synthesized analogs of the initial actives, guided by analysis of active-site interactions,to expand the series and establish SAR and evaluate the ability to solve compound liabilities while maintaining/improvingpotency. We expect two new leads for drug development for TB to be an outcome of this application.b. To perform structural studies of novel Mtb targets for medicinal chemistry and drug discovery.Isocitrate Dehydrogenases: We are proposing to solve the crystal structure of both ICDH enzymes in Mtb. We have successfullyexpressed, purified and crystallized ICDH-1 (Rv3339) and ICDH-2 (Rv0066c), so we expect no major obstacles in solving thestructures. We are also planning to investigate the regulatory system used by Mtb to control ICDH activity by testing both ICDHisoforms against a panel of Ser/Thr kinases, since an inhibition of a regulator of ICDH could also lead to potential growthinhibition. Co-crystal structures and biochemical analysis will be carried out with the inhibitors.PykA: We have cloned, expressed and purified Mtb pyruvate kinase and obtained soluble and catalytically active protein. Wewill work with the SDC to solve this structure. Comparison of the active site of Mtb PykaA with the structure of human pyruvatekinase (1t5a) will help in future design of selective inhibitors. We are planning to study regulation mechanisms of PykA byintermediate metabolites using inhibitors.GcvB: We propose to study the structure and function of GcvB (Rv1832), which binds glycine for the first decarboxylating step.GcvB uses tetrahydrofolate and NADH as co-factors. The P-protein has been solved in T. thermophilus [31]. We haveexpressed the Mtb protein and found it to be soluble, and we are working on obtaining crystals. We will develop an enzymeassay and use it to screen for inhibitors. In addition to co-crystal structures, the inhibitors will be used to probe whether theGCS is needed to detoxify glyoxylate under conditions of fatty acid metabolism.Ndh2: We will determine the crystal structure of Ndh2 to support design of more potent inhibitors. We will express and crystallize the N-terminal extracellular domain (1-370) by truncating the membrane-spanning region (370-400). We willcrystallize the protein in apo form and with known inhibitors for structure determination.GlgE: We plan to determine the structure of GlgE and ligand complexes to advance the design of chemical inhibitors. We haveoptimized a coupled enzyme assay for screening that monitors phosphate release using MESG. The screen will be completedby the Chemical and Genetic Core and inhibitors identified for crystal structure analysis prior to review of the grant.c. Use M. tuberculosis phage tail fibers as targeted drug delivery for Mtb.Like most phages, mycobacteriophages infect and specifically target mycobacteria. Our hypothesis is that the recognitionbetween phages and mycobacteria, characterized by receptors on the bacterial surface and the tip of phage tail fiber, can beused to target nanoparticle encapsulated drugs to Mtb. The phage tail fiber is composed of tail fiber protein and its C-terminalreceptor binding domain is where phage makes the critical contact for bacteria recognition, followed by infection. Hence, thebinding specificity represents a great potential resource in the targeted nanoparticle drug delivery for Mtb.Currently, 4505 strains of mycobacteriophages are identified. Based on the tail morphology, they belong to two phage families:Myoviridae (long contractile tail) and Siphoviridae (long noncontractile tail). Each mycobacteriophage strain has a spectrum ofmycobacterial host and only few of them have been characterized. One strain from each family was selected for our initialdevelopment of targeted drug delivery. The strain of TM4 was selected to target Mtb and the strain of bxz1 was selected totarget M. smegmatis for demonstrating the recognition mechanism and establishing the targeted delivery in the laboratory setup.Tail fiber protein in mycobacteriophage bxz1: We have identified that gp232 is the tail fiber protein in bxz1. The production oftail fiber protein was optimized to obtain soluble and stable protein, containing only the C-terminal receptor binding domain. Wehave determined the tip structure of gp232 and demonstrated the binding specificity of this recombinant gp232 toward M.smegmatis. In order to equip tail fiber protein in the targeted drug delivery system, we are working on optimizing thenanoparticle conjugation to the tail fiber protein.

Progress 07/08/14 to 07/08/19

Outputs
Target Audience:The target audience is very wide and it ranges from the scientific community of faculties, research scientists, students to general public with concerns about diseases like tuberculosis and cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The lab has undergraduate, graduate and post-doctoral fellows being trained every year under this project. How have the results been disseminated to communities of interest?The results are disseminated to communities of interest via journal papers, poster presentations, public presentations, meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the course of this reporting period we were able to identify and purify two mycobacteriophage tail fiber proteins, isolated from phages Bxz1 and TM4. Using fluorescence microscopy, we demonstrated that these purified proteins were able to maintain binding specificity not only between bacteria from different genus, but also between different mycobacterial species. We then designed a prototype microfluidic device that would take advantage of this binding specificity in order to pull down mycobacteria from a diluted sample. This device effectively concentrates mycobacteria in a specific manner, trapping cell from large sample volumes into a single microchannel. We hope that this technology could have some real-life applications in the field of Mycobacterium tuberculosis diagnostics. Indeed, the bacterial load in sputum is often low and can be easily missed by the widespread smear microscopy technique. Our device, used as a processing step before smear microscopy could improve diagnosis rate, especially within individuals with low bacterial load. Our device has shown that it could capture approximately 35% of all cells injected into the device.

Publications


    Progress 10/01/18 to 07/08/19

    Outputs
    Target Audience:The target audience is very wide and it ranges from the scientific community of faculties, research scientists, students to general public with concerns about diseases like tuberculosis and cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The lab has undergraduate, graduate and post-doctoral fellows being trained every year under this project. How have the results been disseminated to communities of interest?The results are disseminated to communities of interest via journal papers, poster presentations, public presentations, meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals?In the upcoming year, we will shift our focus towards a simple and affordable readout. Our technology has shown that it is able to efficiently capture mycobacteria: however, we are currently using fluorescence microscopy to detect and count cells, which isn't affordable enough. Our current approach is to use our device as a way to concentrate cells in a very small volume and use sputum smear microscopy techniques to detect cells. This should hopefully increase the bacterial concentration enough that we will be able to detect the presence of the pathogen even at low bacterial loads.

    Impacts
    What was accomplished under these goals? In the last year, we have made improvements to our microfluidics device, adjusting multiple parameters to optimize the mycobacterial capture efficiency. Using tail fiber proteins and the device, we can capture approximately 40% of all mycobacterial cells in the sample. We have also tested a heterogeneous sample and showed very little decrease in capture efficiency despite a large excess of non-mycobacterial cells. Our device has proven to be effective even at very low bacterial concentration.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tang S, Hicks ND, Cheng YS, Silva A, Fortune SM, Sacchettini JC. Structural and functional insight into the Mycobacterium tuberculosis protein PrpR reveals a novel type of transcription factor. Nucleic Acids Res. 2019 Oct 10;47(18):9934-9949. PMID: 31504787
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Dragset MS, Ioerger TR, Zhang YJ, M�rk M, Ginbot Z, Sacchettini JC, Flo TH, Rubin EJ, Steigedal M. Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness. Sci Rep. 2019 Aug 6;9(1):11394.PMID: 31388080
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Carey AF, Rock JM, Krieger IV, Chase MR, Fernandez-Suarez M, Gagneux S, Sacchettini JC, Ioerger TR, Fortune SM. Correction: TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities. PLoS Pathog. 2019 Jun 4;15(6):e1007846. PMID: 31163081
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Brown EE, Miller AK, Krieger IV, Otto RM, Sacchettini JC, Herman JK. A DNA-Binding Protein Tunes Septum Placement during Bacillus subtilis Sporulation. J Bacteriol. 2019 Jul 24;201(16). pii: e00287-19. PMID: 31160399
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Farhat MR, Freschi L, Calderon R, Ioerger T, Snyder M, Meehan CJ, de Jong B, Rigouts L, Sloutsky A, Kaur D, Sunyaev S, van Soolingen D, Shendure J, Sacchettini J, Murray M. GWAS for quantitative resistance phenotypes in Mycobacterium tuberculosis reveals resistance genes and regulatory regions. Nat Commun. 2019 May 13;10(1):2128. PMID: 31086182
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Crespo RA, Dang Q, Zhou NE, Guthrie LM, Snavely TC, Dong W, Loesch KA, Suzuki T, You L, Wang W, O'Malley T, Parish T, Olsen DB, Sacchettini JC. Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase. J Med Chem. 2019 May 9;62(9):4483-4499. PMID: 31002508
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Ballinger E, Mosior J, Hartman T, Burns-Huang K, Gold B, Morris R, Goullieux L, Blanc I, Vaubourgeix J, Lagrange S, Fraisse L, Sans S, Couturier C, Bacqu� E, Rhee K, Scarry SM, Aub� J, Yang G, Ouerfelli O, Schnappinger D, Ioerger TR, Engelhart CA, McConnell JA, McAulay K, Fay A, Roubert C, Sacchettini J, Nathan C. Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition. Science. 2019 Feb 1;363(6426). pii: eaau8959. Erratum in: Science. 2019 Jun 21;364(6446):. PMID: 30705156
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Miller BK, Hughes R, Ligon LS, Rigel NW, Malik S, Anjuwon-Foster BR, Sacchettini JC, Braunstein M. Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway. Elife. 2019 Jan 3;8. pii: e40063. PMID: 30604681
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Dixit A, Freschi L, Vargas R, Calderon R, Sacchettini J, Drobniewski F, Galea JT, Contreras C, Yataco R, Zhang Z, Lecca L, Kolokotronis SO, Mathema B, Farhat MR. Whole genome sequencing identifies bacterial factors affecting transmission of multidrug-resistant tuberculosis in a high-prevalence setting. Sci Rep. 2019 Apr 3;9(1):5602. PMID: 30944370
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Xia Y, Zhou Y, Carter DS, McNeil MB, Choi W, Halladay J, Berry PW, Mao W, Hernandez V, O'Malley T, Korkegian A, Sunde B, Flint L, Woolhiser LK, Scherman MS, Gruppo V, Hastings C, Robertson GT, Ioerger TR, Sacchettini J, Tonge PJ, Lenaerts AJ, Parish T, Alley M. Discovery of a cofactor-independent inhibitor of Mycobacterium tuberculosis InhA. Life Sci Alliance. 2018 Jun 1;1(3):e201800025. PMID: 30456352
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Chen Q, Shah KN, Zhang F, Salazar AJ, Shah PN, Li R, Sacchettini JC, Wooley KL, Cannon CL. Minocycline and Silver Dual-Loaded Polyphosphoester-Based Nanoparticles for Treatment of Resistant Pseudomonas aeruginosa. Mol Pharm. 2019 Apr 1;16(4):1606-1619. PMID: 30817887
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Salazar AJ, Sherekar M, Tsai J, Sacchettini JC. R pyocin tail fiber structure reveals a receptor-binding domain with a lectin fold. PLoS One. 2019 Feb 5;14(2):e0211432. PMID: 30721244


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:The target audience is very wide and it ranges from the scientific community of faculties, research scientists, students to general public with concerns about diseases like tuberculosis and cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The lab has undergraduate, graduate students and post-doctoral fellows being trained every year under this project. How have the results been disseminated to communities of interest?The results are disseminated to communities of interest via journal papers, poster presentations, public presentations, meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals?Over the next year, we are hoping to test our tail fiber technology as well as our diagnostics device with samples more closely related to clinical samples. We will start with heterogenous bacterial cultures and will then proceed to add other elements commonly found in sputum samples to mimic as closely as possible the clinical samples.

    Impacts
    What was accomplished under these goals? Over the last year, we have developed a prototypediagnostics device based on the Mtb phage tail fiber technology, we have been working on. This device combines microfluidics with tail fiber coated magnetic nanoparticles. We have demonstrated that our device is able to isolate and pull-down whole cells from liquid cultures with a capture rate of approximately 15%. This capture rate was measured with a total cell number injected as low as 100 cells.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Daniel-Wayman S, Abate G, Barber DL, Bermudez LE, Coler RN, Cynamon MH, Daley CL, Davidson RM, Dick T, Floto RA, Henkle E, Holland SM, Jackson M, Lee RE, Nuermberger EL, Olivier KN, Ordway DJ, Prevots DR, Sacchettini JC, Salfinger M, Sassetti CM, Sizemore CF, Winthrop KL, Zelazny AM. Advancing Translational Science for Pulmonary NTM Infections: A Roadmap for Research. Am J Respir Crit Care Med. 2018 Nov 14.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Rittershaus ESC, Baek SH, Krieger IV, Nelson SJ, Cheng YS, Nambi S, Baker RE, Leszyk JD, Shaffer SA, Sacchettini JC, Sassetti CM. A Lysine Acetyltransferase Contributes to the Metabolic Adaptation to Hypoxia in Mycobacterium tuberculosis. Cell Chem Biol. 2018 Dec 20;25(12):1495-1505.e3.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Melief E, Kokoczka R, Files M, Bailey MA, Alling T, Li H, Ahn J, Misquith A, Korkegian A, Roberts D, Sacchettini J, Parish T. Construction of an overexpression library for Mycobacterium tuberculosis. Biol Methods Protoc. 2018;3(1):bpy009.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ellenbarger JF, Krieger IV, Huang HL, Gómez-Coca S, Ioerger TR, Sacchettini JC, Wheeler SE, Dunbar KR. Anion-? Interactions in Computer-Aided Drug Design: Modeling the Inhibition of Malate Synthase by Phenyl-Diketo Acids. J Chem Inf Model. 2018 Oct 22;58(10):2085-2091
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Blanc L, Sarathy JP, Alvarez Cabrera N, OBrien P, Dias-Freedman I, Mina M, Sacchettini J, Savic RM, Gengenbacher M, Podell BK, Prideaux B, Ioerger T, Dick T, Dartois V. Impact of immunopathology on the antituberculous activity of pyrazinamide. J Exp Med. 2018 Aug 6;215(8):1975-1986.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Singh SB, Odingo J, Bailey MA, Sunde B, Korkegian A, OMalley T, Ovechkina Y, Ioerger TR, Sacchettini JC, Young K, Olsen DB, Parish T. Identification of cyclic hexapeptides natural products with inhibitory potency against Mycobacterium tuberculosis. BMC Res Notes. 2018 Jun 28;11(1):416.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ganley JG, Carr G, Ioerger TR, Sacchettini JC, Clardy J, Derbyshire ER. Discovery of Antimicrobial Lipodepsipeptides Produced by a Serratia sp. Within Mosquito Microbiomes. Chembiochem. 2018 Apr 26.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Tiwari D, Park SW, Essawy MM, Dawadi S, Mason A, Nandakumar M, Zimmerman M, Mina M, Ho HP, Engelhart CA, Ioerger T, Sacchettini JC, Rhee K, Ehrt S, Aldrich CC, Dartois V, Schnappinger D. Targeting protein biotinylation enhancestuberculosis chemotherapy. Sci Transl Med. 2018 Apr 25;10(438). pii: eaal1803.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Carey AF, Rock JM, Krieger IV, Chase MR, Fernandez-Suarez M, Gagneux S, Sacchettini JC, Ioerger TR, Fortune SM. TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities. PLoS Pathog. 2018 Mar 5;14(3):e1006939.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Negri A, Javidnia P, Mu R, Zhang X, Vendome J, Gold B, Roberts J, Barman D, Ioerger T, Sacchettini JC, Jiang X, Burns-Huang K, Warrier T, Ling Y, Warren JD, Oren DA, Beuming T, Wang H, Wu J, Li H, Rhee KY, Nathan CF, Liu G, Somersan-Karakaya S. Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis. ACS Infect Dis. 2018 May 11;4(5):771-787.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Lehmann J, Cheng TY, Aggarwal A, Park AS, Zeiler E, Raju RM, Akopian T, Kandror O, Sacchettini JC, Moody DB, Rubin EJ, Sieber SA. An Antibacterial ?-Lactone Kills Mycobacterium tuberculosis by Disrupting Mycolic Acid Biosynthesis. Angew Chem Int Ed Engl. 2018 Jan 2;57(1):348-353.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Chandrasekera NS, Berube BJ, Shetye G, Chettiar S, OMalley T, Manning A, Flint L, Awasthi D, Ioerger TR, Sacchettini J, Masquelin T, Hipskind PA, Odingo J, Parish T. Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB. ACS Infect Dis. 2017 Dec 8;3(12):898-916.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Taira J, Morita K, Kawashima S, Umei T, Baba H, Maruoka T, Komatsu H, Sakamoto H, Sacchettini JC, Aoki S. Identification of a novel class of small compounds with anti-tuberculosis activity by in silico structure-based drug screening. J Antibiot (Tokyo). 2017 Nov;70(11):1057-1064.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Yang K, Chang JY, Cui Z, Li X, Meng R, Duan L, Thongchol J, Jakana J, Huwe CM, Sacchettini JC, Zhang J. Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis. Nucleic acids research. 2017; 45(18):10884-10894.


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:The target audience is very wide and it ranges from the scientific community of faculties, research scientists, students to general public with concerns about diseases like tuberculosis and cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The lab has several graduate students being trained every year under this project. How have the results been disseminated to communities of interest?The results are disseminataed to communities of interest via journal papers, poster presentations, public presentations, meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals?We are currently trying to further utilize our Mtb immobilizing microfluidics device to obtain binding data and improve its efficiency. Ultimately, we would like to have the ability to inject a heterogeneous sample, which would be a clinical sample surrogate, and specifically isolate Mtb cells.

    Impacts
    What was accomplished under these goals? Over the last year, we have done several proof-of-concept experiments to demonstrate the range of possible applications derived from our Mtb tail fiber protein technology. We have evidence that tail fiber proteins aggregate around mycobacterial cells in a microfluidics device, which indicates that tail fiber derived drug delivery could be possible. We were also able to use our tail fiber proteins to specifically immobilize Mtb cell to a microfluidics chamber, which could be used to isolate Mrb from clinical samples improving diagnostics and rapid identification of MDR and XDR isolate by PCR.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Huang HL, Krieger IV, Parai MK, Gawandi VB, Sacchettini JC., Mycobacterium tuberculosis Malate Synthase Structures with Fragments Reveal a Portal for Substrate/Product Exchange. J Biol Chem. 2016 Dec 30;291(53):27421-27432. doi: 10.1074/jbc.M116.750877. Epub 2016 Oct 13.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Puckett S, Trujillo C, Wang Z, Eoh H, Ioerger TR, Krieger I, Sacchettini J, Schnappinger D, Rhee KY, Ehrt S., Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2017 Mar 14;114(11):E2225-E2232. doi: 10.1073/pnas.1617655114. Epub 2017 Mar 6. PMID: 28265055
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Aggarwal A, Parai MK, Shetty N, Wallis D, Woolhiser L, Hastings C, Dutta NK, Galaviz S, Dhakal RC, Shrestha R, Wakabayashi S, Walpole C, Matthews D, Floyd D, Scullion P, Riley J, Epemolu O, Norval S, Snavely T, Robertson GT, Rubin EJ, Ioerger TR, Sirgel FA, van der Merwe R, van Helden PD, Keller P, B�ttger EC, Karakousis PC, Lenaerts AJ, Sacchettini JC., Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13., Cell. 2017 Jul 13;170(2):249-259.e25. doi: 10.1016/j.cell.2017.06.025. Epub 2017 Jun 29. PMID: 28669536
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Pham TV, Murkin AS, Moynihan MM, Harris L, Tyler PC, Shetty N, Sacchettini JC, Huang HL, Meek TD., Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis., Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):7617-7622. doi: 10.1073/pnas.1706134114. Epub 2017 Jul 5. PMID: 28679637


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The structural data obtained as well as the early functional results are currently in manuscript preparation. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on microfluidics experiments, which we believe will demonstrate the ability of the tail fiber proteins to deliver a drug payload or a reporter molecule, for both therapeutics and diagnostics applications. We are also working on solving the crystal structure of the TM4 tail fiber protein as well as the identification the host receptor. We believe gaining structural knowledge at the molecular level would help us better understand the interactions and possibly improve the binding through mutagenesis.

    Impacts
    What was accomplished under these goals? Over the last year we have shown the ability to use tail fiber proteins as a specific targeting molecule. We have isolated and purified 2 tail fibers from different mycobacteriophages; Bxz1 and TM4. We have demonstrated that only the tail fiber isolated from TM4 had the ability to target Mycobacterium tuberculosis, while both could target Mycobacterium smegmatis. This is in agreement with the host range of each mycobacteriophage, correlating the tail fiber's ability to bind the bacteria, with the ability to infect and cause lysis.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Huang HL, Krieger IV, Parai MK, Gawandi VB, Sacchettini JC.Mycobacterium tuberculosis Malate Synthase Structures with Fragments Reveal a Portal for Substrate/Product Exchange.J Biol Chem. 2016 Dec 30;291(53):27421-27432. doi: 10.1074/jbc.M116.750877.
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Palencia A, Li X, Bu W, Choi W, Ding CZ, Easom EE, Feng L, Hernandez V, Houston P, Liu L, Meewan M, Mohan M, Rock FL, Sexton H, Zhang S, Zhou Y, Wan B, Wang Y, Franzblau SG, Woolhiser L, Gruppo V, Lenaerts AJ, O'Malley T, Parish T, Cooper CB, Waters MG, Ma Z, Ioerger TR, Sacchettini JC, Rullas J, Angulo-Barturen I, P�rez-Herr�n E, Mendoza A, Barros D, Cusack S, Plattner JJ, Alley MR. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyle-tRNA Synthetase. Antimicrob Agents Chemother. 2016 Sep 23;60(10):6271-80. doi: 10.1128/AAC.01339-16.
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Mart�nez-Hoyos M, Perez-Herran E, Gulten G, Encinas L, �lvarez-G�mez D, Alvarez E, Ferrer-Bazaga S, Garc�a-P�rez A, Ortega F, Angulo-Barturen I, Rullas-Trincado J, Blanco Ruano D, Torres P, Casta�eda P, Huss S, Fern�ndez Men�ndez R, Gonz�lez Del Valle S, Ballell L, Barros D, Modha S, Dhar N, Signorino-Gelo F, McKinney JD, Garc�a-Bustos JF, Lavandera JL, Sacchettini JC, Jimenez MS, Mart�n-Casabona N, Castro-Pichel J, Mendoza-Losana A. Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor. EBioMedicine. 2016 Jun;8:291-301. doi: 10.1016/j.ebiom.2016.05.006.
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Bhatt VS, Zeng D, Krieger I, Sacchettini JC, Cho JH. Binding Mechanism of the N-Terminal SH3 Domain of Crkll and Proline-Rich Motifs in cAbl. Biophys J. 2016 Jun 21;110(12):2630-41. doi: 10.1016/j.bpj.2016.05.008.


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The structural data obtained as well as the early functional results are currently in manuscript preparation. What do you plan to do during the next reporting period to accomplish the goals?Since our M. smegmatis tail fiber protein showed the ability to specifically target M. smegmatis, we plan to reproduce our results and isolate an Mtb specific tail fiber protein. We are currently working on obtaining the structure of this Mtb tail fiber protein using x-ray crystallography.We are also interested in getting a better understanding of the tail fiber/mycobacteria interaction, and are working on identifying the mycobacterial receptors involved in phage adsorption. Our first approach will be to identify phage infection-resistant mutants to gain a better understanding of which protein or biological pathways might be involved in phage interactions. Lastly we want to demonstrate the ability to deliver antibiotics using mycobacteriophage tail fiber proteins. We have already shown its ability to deliver a fluorophore, we now want to test whether it can deliver larger cargo such as antibiotic-loaded nanoparticles.

    Impacts
    What was accomplished under these goals? In the past year, we have successfully identified and solved the structure of a mycobacteriaphage (Bxz1) tail fiber protein targeting the Mtb homologue Mycobaterium smegmatis. We have also shown that a soluble truncated tail fiber protein can specifically target mycobacteria within a mixed bacterial population.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Dragset MS, Poce G, Alfonso S, Padilla-Benavides T, Ioerger TR, Kaneko T, Sacchettini JC, Biava M, Parish T, Arg�ello JM, Steigedal M, Rubin EJ. A Novel Antimycobacterial Compound Acts as an Intracellular Iron Chelator. Antimicrob Agents Chemother. pii: AAC.05114-14
    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Harshbarger W, Miller C, Diedrich C, Sacchettini J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure;23(2):418-24. doi: 10.1016/j.str.2014.11.017. Epub 2015 Jan 15.


    Progress 07/08/14 to 09/30/14

    Outputs
    Target Audience: The traget audience is very wide and it ranges from the scientific community of faculties, research scientists, students to general public with concerns about diseases like tuberculosis and cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The lab has several undergradaute, graduate and postdoctoral students being trained every year under this project. How have the results been disseminated to communities of interest? The results are disseminated to commuinites of interest via journal papers, poster presenations, public presenations, meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals? We plan to keep on wroking on our projects to progress on our goals and objectives in the next reporting period. We have made considerable progress on several projects. We have a lot of experiments planned.

    Impacts
    What was accomplished under these goals? OUTPUTS: Considerable progress has been made over the last year in our efforts to discover new targets and drugs against Mycobacterium tuberculosis. Using whole-genome sequencing (WGS) of isogenic mutants selected for resistance to various HTS compounds, we have identified several new enzymes in M. tuberculosis as targets of whole-cell active inhibitors. Some of the most interesting targets identified are described below. We have conducted medicinal chemistry and structural-based studies to develop leads that target M. tuberculosis Pks13 and AccD6, both of which are important components in the synthesis of the mycobacterial cell wall. For Pks13 a benzofuran was discovered through a whole-cell screen for molecules with growth inhibition activity against M. tuberculosis H37Rv. For AccD6 the herbicide quizalofop was found to have activity against M. tuberculosis AccD6 as well as cell cultures of M. tuberculosis. To date, we have solved crystal structures of the enzymes in apo form, developed enzyme assays, identified inhibitors, and obtained crystal structures of complexes with whole-cell actives. Medicinal chemistry on these targets have resulted in hundreds of inhibitors with better potency and low toxicity. Mice PK and toxicity studies have shown encouraging results and we are currently carrying out mice efficacy studies. Around 4,000 compounds have been screened against M. tuberculosis Iso-Citrate Dehydrogenase – 2 (ICDH2) via the enzyme assay including two commercially available Human ICDH mutant inhibitors, AGI-6780 and AGI-5198. Four of them have shown inhibition against ICDH2. A higher resolution (2.3Å) ICDH2 structure was obtained. The study of the inhibitor complex structures is in progress. We have performed a screen of over 50,000 drug-like compounds using an assay on New Delhi Metallo β-lactamase (NDM-1) enzyme, the enzyme that causes drug resistance in gram-negative bacteria like Klebsiella. Three hundred forty-four structurally diverse hits have been identified. We have begun to further characterize these hits to learn more about their physical and chemical properties, their ability to bind NDM-1, their toxicity, potency, and pharmacokinetic profiles. Using secondary assays we ensure that hits bind NDM-1. Crystal structures of NDM-1 in complex with inhibitors has been evaluated. Our best compound has already gone through very limited in vivo dose range-finding toxicity studies and preliminary PK. We are poised to begin efficacy trials of our best compounds in a mouse model of NDM-1 positive Klebsiella infection. We have performed structural studies of novel M. tuberculosis targets for medicinal chemistry and drug discovery. PykA (Rv1617) - We have determined the crystal structure of M. tuberculosis pyruvate kinase and have identified the allosteric effector bound to the regulatory site. The regulatory site has been found to be different and the active site is not highly conserved in comparison to human pykA structure. We have screened our library hits for whole cell activity, and found several molecules which inhibit M. tuberculosis pykA in the nM range. We will develop selective inhibitors against M. tuberculosis PykA and co-crystal structures will be solved with the inhibitors bound. After screening 52,174 chemically diverse compounds that would reverse drug-resistance to CHOP in Non-Hodgkin Lymphoma cells, we have discovered an FDA-approved compound for the treatment of an infectious disease. Given its known clinical utility and good toxicological profile, we designed and synthesized over 40 analogs to improve efficacy and pharmacological properties of the parent compound, and identified RTI-79 as our lead compound. RTI-79 has a wide spectrum of activity in many lymphoma and ovarian cancer cell lines and synergizes with multiple chemotherapeutics. Mechanistic studies show that RTI-79 results in rapid increases in ROS and inhibition of intracellular drug efflux. Further, RTI-79 blocks the cellular xenobiotic response by decreasing NRF2 protein levels and decreasing expression of its target genes involved in antioxidant response. Preclinical in vivo studies indicate RTI-79 is both safe and effective. Pharmacokinetic studies show profiles similar to the parent drug and there is no overt toxicity. Significantly, RTI-79 is effective at synergistic tumor growth delay in multiple mouse xenograft models, thus predicting clinical efficacy. OUTCOMES/IMPACT: We have made significant advancements towards solving structures of drug targets from tuberculosis while expanding our work in understanding the mechanisms of action for drugs acting against cancer. We have been involved in solving the structures and characterizing M. tuberculosis targets with unknown functions. In collaboration with the Bill and Melinda Gates Foundation and the NIH, we have developed two lead molecules, active against M. tuberculosis. These molecules are currently undergoing efficacy studies in mice. We are also in talks with Scott and White Hospital to conduct clinical trials on our lead cancer molecule for which we have successfully completed preclinical studies. We have received many compounds from our collaborators and have generated hundreds of compounds via the medicinal chemistry facility for high-throughput screening. We will continue to screen large numbers of compounds to identify inhibitors or a target gene.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Joshua Odingo, Theresa OMalley, Edward A. Kesicki, Torey Alling, Mai Ann Bailey, Julie Early, Juliane Ollinger, Suryakanta Dalai, Naresh Kumar, Ravindra Vikram Singh, Philip A. Hipskind, Jeffrey W. Cramer, Thomas Ioerger, James Sacchettini, Richard Vickers, Tanya Parish, Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents, Bioorganic & Medicinal Chemistry, Volume 22, Issue 24, 15 December 2014, Pages 6965-6979, ISSN 0968-0896, http://dx.doi.org/10.1016/j.bmc.2014.10.007. (http://www.sciencedirect.com/science/article/pii/S0968089614007202) Keywords: Tuberculosis; Mycobacterium tuberculosis; Antibacterial activity; 2,4-Diaminoquinazoline; Dioxygenase
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Manchi C. M. Reddy, Ardala Breda, John B. Bruning*, Mukul Sherekar, Spandana Valluru, Cory Thurman, Hannah Ehrenfeld and James C. Sacchettini Structure, Activity, and Inhibition of the Carboxyltransferase ?-Subunit of Acetyl Coenzyme A Carboxylase (AccD6) from Mycobacterium tuberculosis. 4 August 2014, doi: 10.1128/AAC.02574-13
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Nixon MR, Saionz KW, Koo MS, Szymonifka MJ, Jung H, Roberts JP, Nandakumar M, Kumar A, Liao R, Rustad T, Sacchettini JC, Rhee KY, Freundlich JS, Sherman DR. Folate pathway disruption leads to critical disruption of methionine derivatives in Mycobacterium tuberculosis. Chem Biol. 2014 Jul 17;21(7):819-30. doi: 10.1016/j.chembiol.2014.04.009. Epub 2014 Jun 19.
    • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Wayne Harshbarger, Chase Miller, Chandler Diedrich, and James Sacchettini. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure Journal.
    • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Xiaojun Li, Qingan Sun, Cai Jiang, Kailu Yang, Li-Wei Hung, Junjie Zhang, James C Sacchettini. Structure Insight of Ribosomal Silencing in Mycobacterium tuberculosis. Structure Journal