US008785499B2

(12) United States Patent (10) Patent No.: US 8,785.499 B2 Mackerell, Jr. et al. (45) Date of Patent: Jul. 22, 2014

(54) TARGETING NAD BIOSYNTHESIS IN Medicinal Chemistry Letters 18, 2008, pp. 3932-3937, cited BACTERAL PATHOGENS in ISR. A. K. Halve et al. “N/C-4 substituted azetidin-2-ones: Synthesis and (75) Inventors: Alexander Mackerell, Jr., Baltimore, preliminary evaluation as new class of antimicrobial agents.” MD (US); Hong Zhang, Dallas, TX Bioorganic & Medicinal Chemistry Letters 17, 2007, pp. 341-345, (US); Andrei Osterman, San Diego, CA cited in IRS. P. V. Desai et al. “Identification of Novel Parasitic Cysteine Protease (US); Rohit Kolhatkar, Loves Park, IL Inhibitors. Using Virtual Screening. 1. The ChemBridge Datebase.” (US) Journal of Medicinal Chemistry 2004, No. 47, pp. 6609-6615, cited (73) Assignees: University of Maryland, Baltimore, in ISR. H.J. Yoon et al..."Crystal Structure of Nicotinic Acid Mononucleotide Baltimore, MD (US); The Board of Adenylyltransferase from Pseudomonas aeruginosa in its Apo and Regents of the University of Texas Substrate-complexed Forms Reveals a Fully Open Conformation.” System, Austin, TX (US); Journal of Medicinal Chemistry, 2005, No. 351, pp. 258-265. Sanford-Burnham Medical Research S. Lu et al. “Structure of nicotinic acid mononucleotide Institute, La Jolla, CA (US) adenylyltransferase from Bacillus anthracis,” Structural Biology and Crystalization Communications, 2008, No. 64, pp. 893-898. (*) Notice: Subject to any disclaimer, the term of this H. Zhang et al. "Crystal Structures of E. coli Nicotinate patent is extended or adjusted under 35 Mononucleotide Adenylyltransferase and its Complex with U.S.C. 154(b) by 228 days. Deamido-NAD.” Structure, Vo. 10, Jan. 2002, pp. 69-79. A. M. Olland et al. “Identification, Characterization, and Crystal (21) Appl. No.: 13/383,340 Structure of Bacillus subtilis Nicotinic Acid Mononucleotide Adenylyltransferase.” The Journal of Biological Chemistry, vol. 277. (22) PCT Filed: Jul. 12, 2010 No. 5, Feb. 1, 2002, pp. 3698-3707. S. Han et al. “Crystal Structure of Nicotinic Mononucleotide (86). PCT No.: PCT/US2O10/041708 Adenylyltransferase from Staphyloccocus aureus: Structural Basis S371 (c)(1), for NaAD Interaction in Functional Dimer,” Journal of Mol. Biol., 2006, No. 360, pp. 814-825. (2), (4) Date: Apr. 10, 2012 V. C. Sershon et al. "Kinetic and X-Ray Structural Evidence for Negative Cooperativity in Substrate Binding to Nicotinate (87) PCT Pub. No.: WO2011/006158 Mononucleotide Adenylyltransferase (NMAT) from Bacillus PCT Pub. Date: Jan. 13, 2011 anthracis,” Journal of Mol. Biol. 2009, No. 385, pp. 867-888. International Search Report of PCT/US2010/041708, date of mailing (65) Prior Publication Data Mar. 28, 2011. Written Opinion of PCT/US2010/041708, date of mailing Mar. 28, US 2012/O190708 A1 Jul. 26, 2012 2011. L. Sorcietal. “Targeting NAD Biosynthesis in Bacterial Pathogens: Related U.S. Application Data Structure-Based Development of Inhibitors of Nicotinate Mononucleotide Adenylyltransferase NadD.” Chemistry & Biology (60) Provisional application No. 61/224,504, filed on Jul. 16, Aug. 28, 2009, pp. 849-861. 10, 2009. L. Sorci et al. “Targeting Nad Biosynthesis in Bacterial Pathogens: Structure-Based Development of Inhibitors of Nicotinate (51) Int. Cl. Mononucleotide Adenylyltransferase NadD.” Chemistry & Biology A6 IK3I/65 (2006.01) 16, Aug. 28, 2009, Supplemental Data. (52) U.S. Cl. USPC ...... 514/615: 514/614:564/151 * cited by examiner (58) Field of Classification Search USPC ...... 564/151; 514/614, 615 Primary Examiner — Shailendra Kumar See application file for complete search history. (74) Attorney, Agent, or Firm — Westerman, Hattori, Daniels & Adrian, LLP (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The emergence of multidrug-resistant pathogens necessitates 2007/0037752 A1* 2/2007 Ansorge et al...... 514/18 the search for new antibiotics acting on previously unex 2009/0312363 A1 12/2009 Bradner et al. plored targets. Nicotinate mononucleotide adenylyltrans ferase of the NadD family, an essential enzyme of NAD FOREIGN PATENT DOCUMENTS biosynthesis in most bacteria, was selected as a target for structure-based inhibitor development. To this end, the inven WO 2008091349 A1 T 2008 tors have identified small molecule compounds that inhibit bacterial target enzymes by interacting with a novel inhibi OTHER PUBLICATIONS tory binding site on the enzyme while having no effect on Sorcietal, Chemistry and Biology, 2009, 16(8), 849-861.* functionally equivalent human enzymes. J. Finn et al. “Identification of novel inhibitors of methionyl tRNA synthetase (MetRS) by virtual screening.” Bioorganic & 6 Claims, 18 Drawing Sheets U.S. Patent Jul. 22, 2014 Sheet 1 of 18 US 8,785.499 B2

Figure l

Primary screen on as 1,000,000 in silico compound fibrary: top compounds were selected based on normalized vidWattractive energy

Secondary screen (A) on top 20,000 cmpds against muttiple protein against multiple protein conformations from crystallography Conformations from MD siniations

Top 500 cmpds from (A) and (B) were subjected to chemical clustering with finai :

29 (529) 40

12 active cmpds against both E. coli and B. anthracis Nadds were identified and 3 selected for further study

inhibitor-bahladd crystal structures d U.S. Patent Jul. 22, 2014 Sheet 2 of 18 US 8,785.499 B2

Figure 2

8 MW WW W ------W---- W--W--W W -W W WX MW W-8 W 2 5 - capa class 1 & A Cripd CiaSS. . . 3.

OO - empch class 15 +- ...... WMXWWSM.888 &

baMad D

O 25 50 75 OO 25 50 75 200 eciad) U.S. Patent Jul. 22, 2014 Sheet 3 of 18 US 8,785.499 B2

. U.S. Patent Jul. 22, 2014 Sheet 4 of 18 US 8,785.499 B2

Figure 4

ecado inhibition

0.8 c; X. 0.8 ------, IC50 13 +/- 2 M 0.2

baNad) inhibition

IC50 16+/- 4 M

0 1 2 3 4 50 SO, 0 80 SO M U.S. Patent Jul. 22, 2014 Sheet 5 of 18 US 8,785.499 B2

Figure 5 A

E In 2.5 s one 5 are O O -- 30 a 60 3 - OO ce

B

E 100 O 9. 2. 50 e

s -0.03 0.00 0.03 0.06 0.09 1/NaMN (uM") U.S. Patent Jul. 22, 2014 Sheet 6 of 18 US 8,785.499 B2

Figure 6 A

/

N

3. U.S. Patent Jul. 22, 2014 Sheet 7 of 18 US 8,785.499 B2

F.igure 7

U.S. Patent Jul. 22, 2014 Sheet 8 of 18 US 8,785.499 B2

Figure 8

U.S. Patent Jul. 22, 2014 Sheet 9 of 18 US 8,785.499 B2

Figure 9

U.S. Patent Jul. 22, 2014 Sheet 10 of 18 US 8,785.499 B2

Figure O

U.S. Patent Jul. 22, 2014 Sheet 11 of 18 US 8,785.499 B2

Figure 11

A cir'sh O C B Ors 103 IC50=33M 105 ICsoX200 Mre, 9 t "r's C 04 ICso=25M Bror C. H O C 113 IC50=170M / or " BrorH 9 "10 f 101 IC50=O 4M C 115 IC50=122uM cr's 2 Cso as 25pily --S. Cl R H 111 IC50=30puM C lazy... Cso : 3-33ity. " O

0.

baMadd d ecado 3. 8 8

9. S 4. 0.

82 ,

8 s S8 8 2. 48 s 8 U.S. Patent Jul. 22, 2014 Sheet 12 of 18 US 8,785.499 B2

Figure 12

U.S. Patent Jul. 22, 2014 Sheet 13 of 18 US 8,785.499 B2

Figure 13

U.S. Patent Jul. 22, 2014 Sheet 14 of 18 US 8,785.499 B2

U.S. Patent Jul. 22, 2014 Sheet 15 of 18 US 8,785.499 B2

Figure 15

Scheme 1

HN NH ONN84- ),N-NH O o sea- S. O HN1 O O "C) C -y NH C CS-a 1. 1021 a) benzene-1,4-dicarbaldehyde, ethanol, reflux

Figure 16

Scheme 2

A. O NH2 e-Na1O ames A. O : O-1 H.b oaR^- o

2 3 4. O

o, O 2N ^n HN OH OH x X -N-1 " x .. "2 case O -- 0 O O O O

"Cc C C "Cc 6 7 8

a) water. b) succinic anhydride, DMF, 70°C. c) HBTU, DIPEA, DMF. d) TFACHCl e) Ethanol, 1N NaOH. U.S. Patent Jul. 22, 2014 Sheet 16 of 18 US 8,785.499 B2

Figure 17

Scheme 3

OH HN N OH O2 oS-K)-ONN 4C - powers, " O o " -e-, O )- O HNO O "C N10 C NH a- (-y C 102.2 8 102.3 a) napthalene-l-carbaldehyde, ethanol, reflux. b)benzene-1,4-dicarbaldehyde, ethanol, reflux U.S. Patent Jul. 22, 2014 Sheet 17 of 18 US 8,785.499 B2

Figure 18

KS is as: S S is S S SS is 3S is sis iss:

ississ $issa's U.S. Patent Jul. 22, 2014 Sheet 18 of 18 US 8,785.499 B2

Figure I s

3 & US 8,785,499 B2 1. 2 TARGETING NAD BOSYNTHESIS IN NadD converts NaMN, the first intermediate shared by the BACTERAL PATHOGENS most common de novo and Salvage/recycling routes, to nico tinic acid adenine dinucleotide (NaAD). Therefore, this STATEMENT REGARDING FEDERALLY enzyme should be indispensable in all bacterial species that SPONSORED RESEARCH ORDEVELOPMENT 5 utilize one or both of these routes for NAD biosynthesis. This is consistent with gene essentiality data for a number of This invention was made with the support of the U.S. bacterial species (as reviewed in 3, 16). For example, the government under Grant Number AI059146 from the nadD gene was shown to be essential for Survival in Staphy National Institute of Health (NIH). The U.S. government has lococcus aureus and Streptococcus pneumoniae that are fully certain rights in this invention. 10 dependent on niacin salvage (via PncA-PncB route). It is also essential in Escherichia coli and Mycobacterium tuberculo CROSS REFERENCE TO RELATED sis, organisms that harbor both the de novo (NadB-NadA APPLICATIONS NadC) and the salvage pathways. Remarkably, it has been recently demonstrated that NAD downstream pathway holds This application is a 371 of PCT/US10/41708 Jul. 12, 15 as an attractive target in both actively growing and nonrepli 2010, which claims the benefit of U.S. Provisional Applica cating pathogens 17. NadD is present in nearly all important tion No. 61/224,504 filed Jul. 10, 2009, which is hereby pathogens with only a few exceptional cases, such as Hae incorporated by reference. mophilus influenzae which lacks most of NAD biosynthetic machinery and is dependent on Salvage of the so-called V-fac TECHNICAL FIELD tors 18. Many representatives of the NadD family from pathogenic The invention relates to microbiology. The invention fur and model bacteria have been characterized mechanistically ther relates to methods of treating a microbial infection. In and structurally 19-24. All of these enzymes have a strong further aspects the invention relates to treating a bacterial substrate preference for NaMN over its amidated analog, infection. 25 NMN. On the other hand, all three isoforms of the function ally equivalent human enzyme (hsNMNAT1, hisNMNAT-2 BACKGROUND OF INVENTION and hsNMNAT-3) have an almost equal catalytic efficiency for either substrate, NaMN or NMN 25, 26. The observed The versatility and resourcefulness of microbes in devel difference in substrate specificity reflects the dual physiologi oping resistance to various therapies are widely recognized. 30 cal role of the human enzyme (hereafter referred to as hisN Although chemical modifications of existing drugs and the MNAT) in the adenylation of both intermediates contributing development of novel inhibitors against a handful of previ to NAD biogenesis 7,27. Notably, among the three bacterial ously established targets has proven to be successful in the enzymes of the target pathway, NadD has the lowest sequence short term, it is also apparent that new drug targets need to be similarity to its human counterparts 3. Comparative analy explored to maintain and extend efficacious antibacterial 35 sis of 3D structures of bacterial Nad) and hSNMNAT therapy in the long run 1. The need for new targets is further revealed significant differences between their active site con exacerbated by the emergence of bacterial pathogens with formations 15, which are likely responsible for their distinct natural resistance to existing antibiotics and by a potential Substrate specificities, thus opening an opportunity for selec threat of pathogens with engineered antibiotic resistance. tive targeting. NAD(P) biosynthesis as a promising, albeit relatively 40 It is apparent that there is a need in the art for novel unexplored target pathway for the development of novel anti antimicrobial agents. To this end, the inventors have selected microbial agents 2-4. Cofactors of the NAD pool are indis the NadD enzyme as a target for the development of specific pensable as they are involved in hundreds of redox reactions inhibitors based on a number of criteria Such as essentiality, in the cell. Additionally, NAD is utilized as a cosubstrate by a broad conservation and structure-function distinction from its number of non-redox enzymes (e.g., by bacterial DNA 45 human counterpart. ligases and protein deacetylases of the Cobb/Sir2 family). This dictates the need to maintain NAD homeostasis via its BRIEF SUMMARY OF INVENTION active resynthesis and recycling of NAD degradation prod ucts. Recently, a number of insightful reviews have empha The emergence of multidrug-resistant pathogens necessi sized the potential of NAD(P) biosynthetic enzymes as drug 50 tates the search for new antibiotics acting on previously unex targets for the treatment of cancer, autoimmune diseases, and plored targets. Nicotinate mononucleotide adenylyltrans neurodegenerative disorders 5-8. Although the early steps ferase of the NadD family, an essential enzyme of NAD in NAD biogenesis and recycling vary substantially between biosynthesis in most bacteria, was selected as a target for species, the enzymes driving the downstream conversion of structure-based inhibitor development. Using iterative in nicotinic acid mononucleotide (NaMN) to NAD and NADP 55 silico and in vitro screens, the inventors identified small mol are present in nearly all analyzed bacterial genomes2, 9. ecule compounds that efficiently inhibited target enzymes Therefore, all three enzymes of this pathway NaMN ade from Escherichia coli (ecnadD) and Bacillus anthracis (ba nylyltransferase (EC 2.77.18), NAD synthetase (EC 6.3.1.5) NadD), but which had no effect on functionally equivalent and NAD kinase (EC 2.7.1.23) (encoded by the conserved human enzymes. Importantly, the results of this study for the genes nadD, nadE and nadF, respectively), represent promis 60 first time validated NadD as a drug target for the development ing broad-spectrum antibacterial targets. The observed essen of broad-spectrum antibacterial compound. tiality of the respective genes is due to bacteria being unable The foregoing has outlined rather broadly the features and to uptake phosphorylated pyridine nucleotides 2, 3. Recent technical advantages of the present invention in order that the progress in the development of inhibitors targeting the last detailed description of the invention that follows may be two enzymes, NadE 10-12 and NadF 13, 14, provides 65 better understood. Additional features and advantages of the additional validation of NAD biosynthesis as a target path invention will be described herein, which form the subject of way. the claims of the invention. It should be appreciated by those US 8,785,499 B2 3 4 skilled in the art that any conception and specific embodiment evant side chains are shown as Sticks. Hydrogen bonds are disclosed herein may be readily utilized as a basis for modi shown as dotted lines. Water molecules are shown as small fying or designing other structures for carrying out the same red spheres. C). Surface representation of the inhibitor bind purposes of the present invention. It should also be realized by ing site on baNadD, colored by the electrostatic potentials. those skilled in the art that Such equivalent constructions do Three water molecules adjacent to 1 02 are shown as green not depart from the spirit and scope of the invention as set spheres. forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its FIG. 10. Comparison of the binding modes of 1 02 (ma organization and method of operation, together with further genta), 3 02 (yellow), and the product deamido-NAD(blue). objects and advantages will be better understood from the 10 (A). Superposition of baNadD bound 102 with 3 02 showing following description when considered in connection with the overlapping binding mode. The protein conformations of the accompanying figures. It is to be expressly understood, the two structures are essentially identical and a single ribbon however, that any description, figure, example, etc. is pro diagram is shown. (B). Superposition of the baNadD-1 02 vided for the purpose of illustration and description only and complex (orange) with the baNadD-product complex (cyan). is by no means intended to define the limits the invention. 15 1 02 is in magenta; the product deamido-NAD is in blue. FIG. 11. (A). Structure and activities of representative BRIEF DESCRIPTION OF DRAWINGS Class 1 compounds. The compounds cocrystallized with FIG. 1. Flowchart of the structure-based approach for baNadD are indicated with labels. (B). Dose dependent inhi developing bacterial Nad) inhibitors. bition by compound 1 02 1 against baNadD (left panel) FIG. 2. Correlation analysis of IC50 values for classes 1,3 and ecNadD (right panel). and 15 compounds. The analysis, restricted to compounds FIG. 12. Structure of baNadD-1 02 1 complex. (A). The with IC50 values <0.2 mM, was computed on the assumption 2Fo-Fc map of 1 02 1, the two formate molecules (For)and that both 1050 values for E. coli and B. anthracis Nad)s the Surrounding regions. (B). Superposition of the enzyme follow a Gaussian distribution (Pearson correlation). 25 bound 1 02 1 (blue) with 1 02 in its two orientations FIG. 3. Bacterial growth inhibition. (A) and (B) Effect of (represented in two different shades of gray). C). Detailed inhibitors of class 1 (100 uM) on cell growth as reflected in interactions between 1 02 1 and baNadD residues. changes of the optical density at 600 nm of E. coli AnadA (A) FIG. 13. Structure of baNadD-1 02 3 complex. A). and B. subtilis BSI 68 (B). (C) and (D) On-target inhibition Compound 1 02 3 (blue sticks) binds between two effect. Overexpression of NadD in E. coli AnadA, nadD+ 30 partially or totally suppresses action of the inhibitors 3 15 baNadD monomers (colored cyanand lightcyan), which have (C) and 1 03 (D), resulting in better cell survival. a difference interface from that in the 1 02 1 complex. The FIG. 4. 01 02 01 inhibition. (A) 01 02 01 inhibition two monomers of baNadD in the 1 02 1 complex are col of ecNadD. (B) 01 02 01 inhibition of baNAD. ored light pink with one monomer Superimposed onto the FIG. 5. NadD inhibition by two lead compounds 3 02 and 35 cyan monomer of the 1 02 3 complex. (B). The Fo-Fc omit 1 02. Hyperbolic plots of initial reaction rate (Lmol/mg/ map for 1 02 3. (C). Superposition of the three enzyme min) as a function of NaMN substrate concentration (uM) bound Class/inhibitors showing the common aromatic bind measured at fixed concentration of the ATP substrate (500 ing site as well as differences in the binding mode of each uM) in the presence of varying concentrations of compounds compound. The protein molecules in the 1 02 1 and 3 02 and 1 02 (0-200 uM range). The same data are also 40 1 02, 3 complexes are shown in light pink and cyan, respec presented by a double-reciprocal (Lineweaver-Burk) plot tively. (B). Detailed interactions between 1 02 3 and illustrating mixed-type inhibition. baNadD residues. FIG. 6. Structure of baNadD in complex with inhibitor FIG. 14. Superposition of the baNadD bound 3 02 (yel 3 02 and comparison with product-bound baNadD struc low), 1 02 (magenta), 1 02 1 (blue) and 1 02 3 (green). ture. (A) Interactions between inhibitor and baNadD. Co. 45 The Surface presentation of the enzyme (colored according to traces of baNadD are shown. Protein residues that interact electrostatic potentials) in the 1 02 complex structure is with 3 02 are shown as sticks. Water molecules are shown as shown. The image also includes three nearby water molecules small spheres. (B) Superimposition of baNAD complex observed in the 1 02 complex structure (cyan spheres) and structure with the inhibitor (3 02) bound structure. the formate molecule observed in the 1 02 1 complex FIG. 7. baNadD-3 02 complex tetramer and 3 02 bind 50 structure. The orientation of deamino-NAD" (thin, atom-col ing. (A) Crystal structure of Bacillus anthracis Nad)-3 02 oredlicorice representation) from the product-complex struc complex. Two baNadD dimers are shown. Only one orienta ture (pdb code 3e27) is also shown. tion of the inhibitor 3 02 (in sticks) is shown in each binding site. (B) Overall structure of baNadD dimer (cyan and blue FIG. 15. Scheme 1. Synthesis of compound 1 02 1 subunits) is shown with bound NaAD product. The orienta 55 FIG. 16. Scheme 2. Synthesis of Compound 8 (N-3-(2- tion of this dimer is similar to monomer C. and B in (A). Chloro-phenylcarbamoyl)-propionylhydrazino-acetic acid FIG. 8. Inhibitor 1 02 binds between two monomers of FIG. 17. Scheme 3. Synthesis of 1 02 2 and 1 02 03 baNadD. (A). The Fo-Fc omit map for 1 02. Two 1 02 molecules, colored green and yellow, respectively, each with FIG. 18. NadD inhibition by two lead compounds 3 02 half occupancy are modeled in the density. (B). 1 02 binds at 60 and 1 02 Hyperbolic plots of initial reaction rate (?mol/mg/ a baNadD monomer-monomer interface formed in the crystal min) as a function of NaMN substrate concentration (FM) of the complex. The two baNadD monomers are colored cyan measured at fixed concentration of the ATP substrate and green respectively. FIG. 9. Interactions of 1 02 with baNadD. (A). Ribbon (500 fM) in the presence of varying concentrations of com representation of baNadD-1 02 complex. Inhibitor 1 02 is 65 pounds 3 02 and 1 02 (0-200? Mrange). The same data are shown as sticks. (B). Detailed interactions between 1 02 and also presented by a double-reciprocal (Lineweaver-Burk) baNadD residues. The C trace of the protein is shown; rel plot illustrating mixed-type inhibition. US 8,785,499 B2 5 6 FIG. 19. Structural basis for selective targeting of bacterial for each species. Energies in kcal/mol. Most favorable energy Nad) for each compound is highlighted in light gray and the least (A) Superposition of baNadD-302 complex (magenta) favorable in dark gray. with apo ecNadD (wheat). Inhibitor 302 is shown as magenta Table 6. Attractive van der Waals inhibitor-protein interac Sticks. 5 tion energies using selected compounds. Values are based on (B) Superposition of baNadD-3 02 complex (magenta) the most favorable attractive vaW energy for each compound with apo human NMNAT-i (blue). Selected residues in over the crystal structures used for docking for each species. baNadD that are involved in inhibitor binding (M109, Y112 Energies in kcal/mol. Most favorable energy for each com and W116) are displayed as thin lines. Corresponding resi pound is highlighted in light gray and the least favorable in 10 dark gray. dues in hsNMNAT-i (L159, S162 and W169) are also shown. Table 7. Crystal Data and refinement statistics. The structure comparison illustrates that the conformations "Rs, X, XII-sl-l/XXIII. "Roxie/Fa-Fasik, Fl. ofbacterial NadD enzymes are very similar around the inhibi where F and F are the observed and calculated structure tor binding region while the human enzyme is more diver factors, respectively. Five percent randomly selected reflec gent.hsNMNAT-1 residues corresponding to baNadIDW116 tions were excluded from refinement and used in the calcu andY112 (W169 and S162, showninthin blue line in B would lation of R. clash with the inhibitor in its present pose. Table 8. Inhibition data for compound primary testing. FIG. 20. Antibacterial assay: on-target versus off-target Compounds were originally selected from an ~million com activity pound library. Antibacterial activity of selected compounds at 100 tM on Table 9. Inhibition data for selected compounds class 1 , E. coli overexpressing NadD compared to a control E. coli 3 and 15 . Inhibition 96 was measured at compound con strain (see Methods for details). The error bars represent the centration of 100 uM for E. coli NadD and 50 uM for B. standard deviation between triplicate samples. anthracis NadD. 1050 values, when applicable, are indicated. FIG. 21. Antibacterial assay: on-target versus off-target Table 10. Selected structures for compounds of class 1 . activity 25 Table 11 Selected structures for compounds of class 3 . Antibacterial activity of selected compounds at 100 uM on Table 12. Additional structures for compounds of class 1 . E. coli overexpressing NadD (blue) compared to a control E. Table 13. Chemical structures of two classes of bacterial coli strain (red) (see Methods for details). The error bars NadD inhibitors as represented by compounds 1 02 and represent the standard deviation between triplicate samples. 3 O2 Table 1. Inhibitory parameters of representative com 30 Table 14. Crystal Data and refinement statistics pounds from two chemotypes. The apparent values of inhibi tory parameters (Ki and C) of two compounds (3 02 and DETAILED DESCRIPTION OF THE INVENTION 1 02) were determined for both enzymes by fitting the kinetic data to the general equation for the mixed-model I. Definitions inhibition (43). The data were collected by varying the 35 concentration of an inhibitor and one of the two substrates Unless otherwise noted, technical terms are used according (NaMN or ATP) at fixed concentration of another substrate to conventional usage. Definitions of common terms in (0.5 mM ATP or NaMN). molecular biology may be found, for example, in Benjamin Table 2. Inhibition of target enzymes and antibacterial Lewin, Genes VII, published by Oxford University Press, activity of selected compounds. 40 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Ency "Inhibitory efficiency of selected compounds (representa clopedia of Molecular Biology, published by Blackwell Pub tive of classes 1, 3, and 15) for two target enzymes, ecnadD lishers, 1994 (ISBN 0632021829); and Robert A. Meyers and baNadD is illustrated by ICso values. Antibacterial (ed.), Molecular Biology and Biotechnology: a Comprehen activity of the same compounds against Gram-negative (E. sive Desk Reference, published by Wiley, John & Sons, Inc., coli) and Gram-positive (B. subtilis, B. anthracis) model spe 45 1995 (ISBN 0471186341); and other similar technical refer cies is reflected by MICs values (the lowest concentration of CCCS, compound causing more than 50% growth inhibition). Only As used herein, 'a' or 'an' may mean one or more. As used single-point high estimates of MICso values were determined herein when used in conjunction with the word "comprising.” (70% growth inhibition at 100 microM for E. coli, and 96% the words “a” or “an' may mean one or more than one. As inhibition at 50 microM for B. subtilis) for a representative of 50 used herein "another may mean at least a second or more. the class 15 that displayed mostly off-target antibacterial Furthermore, unless otherwise required by context, singular activity in E. coli model; NA, not assayed. “MICso of cmpd terms include pluralities and plural terms include the singular. 1 03 for B. anthracis was determined using a different set of As used herein, “about” refers to a numeric value, includ concentrations (120, 60, 30, 15, 7.5, and 3.5 microM) ing, for example, whole numbers, fractions, and percentages, Table 3. Proteins targeted and the identification of residues 55 whether or not explicitly indicated. The term “about gener adjacent to the sphere sets used to direct docking in each ally refers to a range of numerical values (e.g., +/-5-10% of protein. the recited value) that one of ordinary skill in the art would Table 4. Docking energies using selected compounds. Val consider equivalent to the recited value (e.g., having the same ues represent the most favorable energy for each compound function or result). In some instances, the term “about may over the crystal structures used for docking for each species. 60 include numerical values that are rounded to the nearest sig Energies in kcal/mol. Most favorable energy for each com nificant figure. pound is highlighted in light gray and the least favorable in As used herein, “treat' and all its forms and tenses (includ dark gray. ing, for example, treating, treated, and treatment) can refer to Table 5. Electrostatic and van der Waals inhibitor-protein therapeutic or prophylactic treatment. In certain aspects of the interaction energies using selected compounds. Values are 65 invention, those in need thereof of treatment include those based on the most favorable electrostatic or vdW energy for already with a pathological condition of the invention (in each compound over the crystal structures used for docking cluding, for example, a bacterial infection), in which case US 8,785,499 B2 7 8 treating refers to administering to a subject (including, for indicate that the level of structural conservation in the active example, a human or other mammal in need of treatment) a sites of divergentrepresentatives of the NadD family provides therapeutically effective amount of a composition so that the a potential for developing broad-spectrum inhibitors. At the Subject has an improvement in a sign or symptom of a patho same time, the three selected chemotypes showed no appre logical condition of the invention. The improvement may be ciable activity against human counterparts (hsNMNAT-1-3). any observable or measurable improvement. Thus, one of This finding validated another premise of the invention, that skill in the art realizes that a treatment may improve the the distinction between bacterial and human enzymes is Suf patient’s condition, but may not be a complete cure of the ficient for the development of selective (bacterial-specific) pathological condition. In other certain aspects of the inven Nad) inhibitors. tion, those in need thereof of treatment include, those in 10 The essentiality of the nadD gene previously established by which a pathological condition of the invention is to be pre genetic techniques, by itself, does not guarantee that inhibi vented, in which case treating refers to administering to a tion of the NadD enzyme in the cell is possible and may Subject atherapeutically effective amount of a composition to indeed suppress bacterial growth. Moreover, the antibacterial a subject at risk of developing a pathological conditional of activity of the analyzed compounds observed in Gram-nega the invention. 15 tive (E. coli) and Gram-positive (B. subtilis) model systems, while being encouraging, could be due to some effects other II. The Present Invention than inhibition of the NadD enzyme. An E. coli model system to test whether the observed growth suppression was indeed In earlier studies the inventors have used a comparative due to the “on-target action of representative NadD inhibi genomics approach to identify NAD cofactor biosynthesis as tors was used. As illustrated in FIGS. 3C and 3D and FIG.20, a target pathway for development of new anti-infective thera overexpression of the target nadD gene Substantially pies 2, 3). The NadD enzyme was chosen as one of the most increased resistance of this strain toward compounds of attractive targets within this pathway due to its nearly univer classes 1 and 3 . These data directly validated the Nad) sal conservation in bacterial pathogens and its essentiality enzyme as a drug target amenable to inhibition in a bacterial directly confirmed in a number of model bacteria 3. A 25 cell, which results in growth suppression. comparative enzymatic and structural analysis revealed Sub Finally, it was important to test the binding mode of NadD stantial differences between bacterial enzymes and their inhibitors. This seemed particularly important as the steady human counterparts, opening an opportunity for development state kinetic analysis of the representative compounds of both of selective NadD inhibitors. The fact that no drugs are known classes 1 and 3 revealed a mixed-type inhibition with a to act on NadD further contributes to this choice of a target in 30 strong noncompetitive component (FIG. 5). To assess inhibi the context of the growing challenge of multidrug-resistant torbinding mode(s) and to obtain a basis for rational improve bacterial pathogens. ment of the inhibitors, the inventors attempted co-crystalliza In the instant invention, an integrated structure-based tion of both bacterial NadD enzymes with a panel of approach was employed to identify Small-molecule com compounds of classes 1 and 3 . The structure of baNadD in pounds that selectively inhibit enzymes of the Nad) family 35 complex with the compound 3 02 reported here confirmed with a potential broad spectrum of antibacterial activity. that this inhibitor indeed binds in the active site area partially Combining computational screening of a virtual compound overlapping with the targeted NaMN substrate binding site library with experimental testing of inhibitory and antibacte (FIG. 6). Moreover the conformation of the baNadD active rial activity of selected compounds and their analogs, the site in this complex is drastically different from its product inventors have identified and characterized at least two 40 bound conformation. In fact, the inhibitor binding appears to classes (including 3 class of compounds, 1 class of com stabilize the baNadD conformation in its apo form, incom pounds; see Table 2) of inhibitors with distinct chemical patible with substrate binding and catalysis 21, 24. Inhibitor scaffolds (chemotypes) possessing a number of desired prop interference at the level of substrate binding and the stabili erties. Zation of alternative enzyme conformation may provide a The approach of in silica Screening was based on selective 45 rationale for the observed complex (mixed-type) kinetics of targeting of those active site residues that are highly con inhibition. Although the actual inhibitory mechanism is not served among bacterial Nad) enzymes, yet quite distinct fully clear, the obtained structural information is useful for from the human counterpart enzymes 15, 19. A focused further inhibitor optimization via structure-based design and targeting of a nicotinosyl-binding (as opposed to adenosyl synthesis of analogs. For example, engineering additional binding) site was also aimed to exploit the functional differ 50 functional groups that may form specific hydrogen-bond ences between the NaMN-preferring bacterial NadD and interactions with the enzyme may enhance the binding affin human enzymes with dual specificity for NaMN and NMN ity of the compound. substrates 25, 26. The inventors also took advantage of the There is an unmet need in the medical arts related to treat large conformational differences between the apo and Sub ing bacterial infections for which the instant inventions fills a strate-bound enzymes by specifically targeting the enzyme 55 Void. In particular, bacterial resistance was a consideration by active site in the apo form so that the inhibitors would stabi the inventors. An example of bacterial resistance includes lize the enzyme in a catalytically impaired conformation. strains of Staphylococcus aureus resistant to methicillin and The results of the invention support the efficiency and other antibiotics that are becoming more common place. effectiveness of this strategy. First, it observed that an appre Infection with methicillin-resistant S. aureus (MRSA) strains ciable correlation between inhibitory properties of com 60 is also increasing in non-hospital settings. Vancomycin is an pounds against two divergent members of the NadD family, effective treatment for MRSA infections. A particularly trou from Gram-negative (ecNadD) and Gram-positive bacteria bling observation is that S. aureus strains with reduced Sus (baNadD), even at the level of the primary experimental test ceptibility to Vancomycin have emerged recently in Japan and ing of-300 compounds. This trend was even more apparent in the United States. The emergence of Vancomycin-resistant the comparison of inhibitory properties of analogs of the three 65 strains would present a serious problem for physicians and class of compounds (1 ., 3 , and 15 ) selected for detailed patients. Another example of bacterial resistance is illustrated characterization (FIG. 2 and Table 8). These observations in the increasing reliance on Vancomycin, which has led to the US 8,785,499 B2 10 emergence of Vancomycin-resistant enterococci (VRE), bac Brachybacterium, Brachymonas, Brachyspira, Brackiella, teria that infect wounds, the urinary tract and other sites. Until Bradyrhizobium, Branhamella, Brenneria, Brevibacillus, 1989, such resistance had not been reported in United States Brevibacterium, Brevigemma, Brevundimonas, Brochothrix, hospitals. By 1993, however, more than 10 percent of hospi Brucella, Bryantella, Budvicia, Bulleidia, Burkholderia, tal-acquired enterococci infections reported to the Centers for 5 Buttiauxella, Butyribacterium, Butyrivibrio, Byssovorax, Disease Control (“CDC) were resistant. Yet another example Caenibacterium, Caldanaerobacter; Calderobacterium, Cal is apparent when considering Streptococcus pneumoniae dicellulosiruptor, Caldilinea, Caldithrix, Caldocellum, causes thousands of cases of meningitis and pneumonia, as Caloramator, Caloranaerobacter, Caminibacillus, Camini well as 7 million cases of ear infection in the United States bacter; Caminicella, Campylobacter, Capnocytophaga, Car each year. Currently, about 30 percent of S. pneumoniae iso 10 bophilus, Carboxydibrachium, Carboxydocella, Carboxy lates are resistant to penicillin, the primary drug used to treat dothermus, Cardiobacterium, Carnobacterium, this infection. Many penicillin-resistant strains are also resis Caryophanon, Caseobacter; Castellaniella, Catellatospora, tant to other antimicrobial or antibacterial compounds. These Catellibacterium, Catenibacterium, Catenococcus, Catenu examples, as well as many more, Support the notion that there loplanes, Catenulospora, Caulobacter; Cedecea, Cellulomo is a tremendous need in the medical arts for novel antibacte 15 nas, Cellulophaga, Cellulosimicrobium, Cellvibrio, Centi rial compounds. peda, Cerasibacillus, Chainia, Chelatobacter, In certain aspects of the invention, a compound disclosed Chelatococcus, Chitinihacter, Chitinophaga, Chlorobacu herein is useful for treating a bacterial infection. A bacterial lum, Chlorobium, Chloroflexus, Chondrococcus, Chondro infection is an infection that is, in-whole or in-part, caused by, myces, Chromatium, Chronobacterium, Chromohalobacter, for example, exposure to a bacterium from a bacterial genera Chlyseobacterium, Chryseomonas, Chrysiogenes, Citrei and any species or derivative associated therewith, including cella, Citricoccus, Citrobacter; Clavibacter; Clavispo for example, any one or more of the following bacterium rangium, Clostridium, Cobetia, Cohnella, Collimonas, Col genera: Abiotrophia, Acaricomes, Acetitomaculum, Acetiv linsella, Colwellia, Comamonas, Conchiformibius, ibrio, Acetobacter, Acetobacterium, Acetobacteroides, Conexibacter, Coprothermobacter; Coral lococcus, Corio Acetogenium, Acetohalobium, Acetomicrobium, Acetomo 25 bacterium, Corynebacterium, Couchioplanes, Crossiella, nas, Acetonema, Achronobacter, Acidaminobacter, Acidami Cryobacterium, Cryptanaerobacter; Cryptobacterium, nococcus, Acidimicrobium, Acidiphilium, Acidithiobacillus, Cryptosporangium, Cupriavidus, Curtobacterium, Curvi Acidobacterium, Acidocaldus, Acidocella, Acidomonas, Aci bacter; Cyclobacterium, Cystobacter, Cytophaga, Dacty dovorax, Acinetobacter, Acrocarpospora, Actinacidiphilus, losporangium, Dechloromonas, Dechlorosoma, Deferrib Actinoacidiphilus, Actinoalloteichus, Actinobacillus, Actino 30 acter, Defluvihacter, Dehalohacter, Dehalospirillum, baculum, Actinobifida, Actinobispora, Actinocatenispora, Deinohacter, Deinococcus, Deleva, Delfia, Demetria, Den Actinocoralia, Actinokineospora, Actinomadura, Actinomy drosporobacter, Denitrovibrio, Dermabacter, Dermacoccus, ces, Actinoplanes, Actinopolyspora, Actinopycnidium, Acti Dermatophilus, Derxia, Desemzia, Desulfacinum, Desulfar nosporangium, Actinosynnema, Actinotelluria, Adhaerib culus, Desulfatibacillum, Desulfitobacterium, Desulfoarcu acter, Aequori vita, Aerobacter, Aerococcus, Aeronicrobium, 35 lus, Desulfobacca, Desulfobacter; Desulfobacterium, Des Aeromonas, Aestuariibacter, Afipia, Agarbacterium, Agito ulfobacula, Desulfobulbus, Desulfocapsa, Desulfocella, coccus, Agreia, Agrobacterium, Agrococcus, Agromonas, Desulfococcus, Desulfo?aba, Desulfoffigus, Desulfo?iustis, Agromyces, Ahrensia, Albidovulum, Alcaligenes, Alcanivo Desulfohalobium, Desulfomicrobium, Desulfomonas, Des rax, Algibacter, Algoriphagus, Alicycliphilus, Alicyclobacil ulfomonile, Desulfonusa, Desulfonatronovibrio, Des lus, Alishewanella, Alistipes, Alkalibacillus, Alkalibacter; 40 ulfonatronium, Desulfonauticus, Desulfonema, Des Alkalibacterium, Alkalilimnicola, Alkalispirillum, Alkanindi ulfonispora, Desulforegula, Desulforhabdus, ges, Allisonella, Allobaculum, Allochromatium, Allofustis, Desulforhopalus, Desulfosarcina, Desulfospira, Desulfos Alteromonas, Alysiella, Aminobacter; Aminobacterium, Ami porosinus, Desulfotalea, Desulfothermus, Desulfotignum, nomonas, Ammonifex, Ammoniphilus, Amoebobacter, Amor Desulfotomaculum, Desulfovihrio, Desulfovirga, Des phosphorangium, Amphibacillus, Ampullariella, Amycolata, 45 ulfitrella, Desulfurobacterium, Desulfuromonas, Desulfuro Amycolatopsis, Anaeroarcus, Anaerobacter; Anaerobaculum, musa, Dethiosulfovibrio, Devosia, Dialister, Diaphorobacter, Anaerobiospirillum, Anaerobranca, Anaerocellum, Anaero Dichelobacter, Dichotomicrobium, Dickeya, Dictyoglomus, coccus, Anaerofilum, Anaerofustis, Anaerolinea, Anaero Dietzia, Diplococcus, Dokdoa, Dokdonella, Dokdonia, musa, Anaerophaga, Anaeroplasma, Anaerosinus, Anaerosti Dolosicoccus, Donghaeana, Dorea, Duganella, Dyado DeS, Anaerotruncus, Anaerovibrio, AnaerovOrax, 50 bacter, Dyella, Eberthella, Ectothiorhodospira, Edwards Ancalomicrobium, Ancylobacter; Aneurinibacillus, Angio iella, Eggerthella, Eikenella, Elizabethkiingia, Elytrospo coccus, Angulomicrobium, Anoxybacillus, Antarctobacter; rangium, Empedobacter, Enhygromyxa, Ensifer, Aquabacter, Aquabacterium, Aquamicrobium, Aquaspiril Enterobacter, Enterococcus, Enterovibrio, Epilithonimonas, lum, Aquicella, Aquifex, Aquiflexum, Aquinonas, Arachnia, Eremococcus, Erwinia, Erysipelothrix, Erythrobacter, Eryth Arcanobacterium, Archangium, Arcicella, Arcobacter, Areni 55 romicrobium, Erythromonas, Escherichia, Eubacterium, bacter, Arhodomonas, Arizona, Arsenicicoccus, Arsenopho Ewingella, Excellospora, Exiguobacterium, Faecalibacte nus, Arthrobacter; Asanoa, Asiosporangium, Asticcacaulis, rium, Faenia, Falcivibrio, Ferrimonas, Ferrobacillus, Fervi Atopobium, Atopococcus, Atopostipes, Aurantimonas, dobacterium, Filibacter, Filifactor, Filobacillus, Filomicro Aureobacterium, Avibacterium, Axonoporis, Azoarcus, AZO bium, Finegoldia, Flammeovirga, Flavimonas, hydromonas, Azomonas, Azomonotrichon, Azorhizobium, 60 Flavobacterium, Flectobacillus, Flexihacter, Flexistipes, Azorhizophilus, Azospira, AZOspirillum, Azotobacter, Bacil Flexithrix, Fluoribacter, Fluviicola, Formivibrio, Fran lus, Bacterionema, Bacteriovorax, Bacterium, Bacteroides, cisella, Frankia, Frateuria, Friedmanniella, Frigoribacte Balnearium, Balneatrix, Bartonella, Baellovibrio, Beggia rium, Fulvimarina, Fulvimonas, Fundibacter, Fusibacter, toa, Beijerinckia, Belliella, Belnapia, Beneckea, Bergeriella, Fusobacterium, Gaetbulibacter; Gaetbulimicrobium, Betabacterium, Beutenbergia, Bifidobacterium, Bilophila, 65 Gaffkya, Gallibacterium, Gallicola, Garciella, Gardnerella, Blastobacter, Blastochloris, Blastococcus, Blastomonas, Gariaella, Gellidibacter; Gelria, Gemella, Gemmata, Gem Blastopirellula, Bogoriella, Bordetella, Borrelia, Bosea, matimonas, Gemmobacter, Geobacillus, Geobacter, Geoder US 8,785,499 B2 11 12 matophilus, Geopsychrobacter, Georgenia, Geospirillum, trum, Octadecabacter, Odontomyces, Oenococcus, Oerisk Geothermobacter, Geothrix, Geovibrio, Giesbergeria, Gilli ovia, Oleiphilus, Oleispira, Oligella, Oligotropha, Olsenella, sia, GlacieCola, Globicatella, Gluconacetobacter, Glu Opitutus, Orenia, Oribacterium, Ornithinicoccus, Ornithin conoacetobacter, Gluconobacter, Glycomyces, Goodfel imicrobium, Ornithobacterium, Ottowia, Oxalicibacterium, lowia, Gordona, Gordonia, Gracilibacillus, Granulicatella, Oxalobacter, Oxalophagus, Oxobacter, Paenibacillus, Palud Granulobacter, Grimontia, Guggenheimella, Gulosihacter; ibacter, Pandoraea, Pannonibacter, Pantoea, Papillibacter, Haemophilus, Hafinia, Hahella, Halanaerobacter; Hala Paracoccus, Paracolobactrum, Paralactobacillus, Parallio naerohium, Haliangium, Haliscomenobacter, Haloanaero bacillus, Parascardovia, Parasporobaaerium, Parvibaculum, bacter, Haloanaerobium, Halobacillus, Halobacteroides, Parvopolyspora, Pasteurella, Pasteuria, Patulibacter; Pauci Halocella, Halochromatium, Halococcus, Haloincola, 10 hacter; Paucimonas, Pectinatus, Pectobacterium, Pediococ Halolacti bacillus, Halomonas, Halonatronium, Halorho cus, Pedohacter, Pelczaria, Pelobacter, Pelodictyon, Pelomo do spira, Halothermothrix, Halothiobacillus, Halovibrio, nas, Pelospora, Pelotomaculum, Peptococcus, Peptoniphilus, Hellcococcus, Helicobacter, Heliobacillus, Heliobacterium, Peptostreptococcus, Peredibacter, Persephonella, Persi Heliophilum, Heliorestis, Herbaspinllum, Herbidospora, civirga, Persicobacter, Petrimonas, Petrobacter, Petrotoga, Herpetosiphon, Hespellia, Hippea, Hirschia, Hoeflea, Hold 15 Phaeobacter, Phaeospirillum, Phascolarctobaaerium, Phe emania, Holophaga, Hongiella, Hordeomyces, Hvalangium, nylobacterium, Phocoenobacter, Photobacterium, Photo Hydrocarboniphaga, Hydrogenivirga, Hydrogenobacter, rhabdus, Phyllobacterium, Phytomonas, Pigmentiphaga, Hydrogenobaculum, Hydrogenomonas, Hydrogenophaga, Pilimelia, Pimeliobacter, Pirella, Pirellula, Planctomyces, Hydrogenophilus, Hydrogenothermophilus, Hydrogenother Planifiulum, Planobispora, Planococcus, Planomicrobium, mus, Hydrogenovibrio, Hvlemonella, Hymenobacter; Planomonospora, Planopolyspora, Planotetraspora, Planti Hyphonicrobium, Hyphomonas, Idiomarina, Ignavigranum, bacter; Pleomorphomonas, Plesiocystis, Plesiomonas, Ilvobacter, Inflabilis, Inquilinus, Intrasporangium, Iodoba Podangium, Polaribacter, Polaromonas, Polyangium, Poly cier; Isobaculum, Isochromatium, Isoptericola, Jahnia, Jani morphosphora, Pontibacillus, Porphyrobacter, Porphyromo bacter, Jannaschia, Janthinobacterium, Jensenia, Jeotgalic nas, Pragia, Prauserella, Prevotella, Proactinomyces, Pro occus, Jiangella, Jonesia, Kangiella, Kerstersia, 25 micromonospora, Promyxobacterium, Propionibacter, Kibdellosporangium, Kibdelosporangium, Kineococcus, Propionibacterium, Propionicimonas, Propioniferax, Propi Kineosphaera, Kineosporia, Kingella, Kitasatoa, Killas Onigenium, Propiomimicrobium, Propionispira, Propion alospora, Kitasatosporia, Klebsiella, Kluyvera, Knoelia, ispora, Propionivibrio, Prosthecobacter, Prosthecochloris, Kocuria, Kofleria, Koserella, Kozakia, Kribbella, Kurthia, Prosthecomicrobium, Protaminobacter, Proteiniphilum, Pro Kutzneria, Kytococcus, Labrys, Laceyella, Lachnobacterium, 30 teus, Protomonas, Providencia, Pseudaminobacter, Lachnospira, Lactobacillus, Lactobacterium, Lactococcus, Pseudoalteromonas, Pseudoamycolata, Pseudobutyrivibrio, Lactosphaera, Lamprocystis, Lampropedia, Laribacter; Lau Pseudoclavibacter. Pseudomonas, Pseudonocardia, Pseudo tropia, Leadbetterella, Lebetimonas, Lechevalieria, Lecler ramibacter, Pseudorhodobacter, Pseudospirillum, Pseudox cia, Leeuwenhoekiella, Legionella, Leifsonia, Leisingera, anthomonas, Psychrobacter, Psychroflexus, Psychromonas, Leminorella, Lentibacillus, Lentzea, Leptospirillum, Lepto 35 Psychroserpens, Pusillimonas, Pyxicoccus, Ouadrisphaera, thrix, Leptotrichia, Leucobacter; Leuconostoc, Leucothrix, Rahnella, Ralstonia, Ramibacterium, Ramlibacter, Raoul Levilinea, Levinea, Limnobacter; List, Listeria, Listonella, tella, Rarobacter, Rathavibacter, Reinekea, Renibacterium, Loktanella, Lonepinella, Longispora, Lophomonas, Lucibac Renobacter; Rhabdochromatium, Rheinheimera, Rhizo terium, Luteibacter; Luteinonas, Luteococcus, Lysobacter; bacter; Rhizobium, Rhizomonas, Rhodobacter; Rhodobium, Macrococcus, Macromonas, Magnetospirillum, Mahella, 40 Rhodoblastus, Rhodocista, Rhodococcus, Rhodocyclus, Malikia, Malonomonas, Mannheimia, Maribacter; Maricau Rhodoferax, Rhodomicrohium, Rhodopila, Rhodoplanes, lis, Marichromatium, Marinibacillus, Marinilabilia, Marin Rhodopseudomonas, Rhodospirillum, Rhodothalassium, ilactibacillus, Marinithermus, Marinitoga, Marinobacter, Rhodothermus, Rhodovibrio, Rhodovulum, Riemerella, Marinobacterium, Marinococcus, Marinomonas, Marino Rikenella, Robiginitalea, Roseateles, Roseburia, Roseiflexus, spirillum, Marinovum, Marmoricola, Massilia, Megamonas, 45 Roseinatronobacter; Roseobacter; Roseococcus, Roseospira, Megasphaera, Meiothermus, Melittangium, Mesonia, Meso Roseospirillum, Roseovarius, Rothia, Rubritepida, Rubriv philobacter, Mesorhizobium, Methanomonas, Methylobacil ivax, Rubrobacter, Ruegeria, Ruminobacter, Ruminococcus, lus, Methylobacterium, Methylocapsa, Methylocella, Methy Saccharibacter, Saccharococcus, Saccharomonospora, Sac lomicrohium, Methylomonas, Methylophaga, Methylophilus, charophagus, Saccharopolyspora, Saccharothrix, Sagittula, Methylopila, Methylosarcina, Methylotenena, Methylovorus, 50 Salana, Sallegentibacter; Salibacillus, Salinibacter, Salini Microbacterium, Microbispora, Microhulhifer, Micrococcus, bacterium, Salinicoccus, Salinimonas, Salinispora, Salinivi Microcyclus, Microechinospora, Microellobosporia, hrio, Salinospora, Salipiger; Salmonella, Samsonia, San Microlunatus, Micromonas, Micromonospora, Micropo/ guihacter; Saprospira, Sarcina, Sarraceniospora, Scardovia, yspora, Microprulina, Microscilla, Microsphaera, Micros Schineria, Schlegelella, Schwartzia, Sebekia, Sedimenti treptospora, Microtetraspora, Microvirgula, Millisia, Mima, 55 bacter; Segniliparus, Seinonella, Sejongia, Selenomonas, Mitsuokella, Mobiluncus, Modestobacter, Moellerella, Mogi Seliberia, Serinicoccus, Serpulina, Serratia, Shewanella, bacterium, Moorella, Moraxella, Moraxella, (Branhamella), Shigella, Shinella, Shuttleworthia, Silanimonas, Silicibacter, Moraxella, (Moraxella), Morganella, Moritella, Muricauda, Simonsiella, Simplicispira, Simsoniella, Sinorhizobium, Muricoccus, Myceligenerans, Mycetocola, Mycobacterium, Skermania, Slackia, Smaragdicoccus, Smithella, Sodalis, Mycoplana, Myroides, Myxococcus, Nakamurella, Nanno 60 Soehngenia, Sorangium, Sphaerobacter, Sphaerophorus, cystis, Natroniella, Natronincola, Nautilia, Naxibacter, Neis Sphaerosporangium, Sphaerotilus, Sphingobacterium, Sph seria, Nereida, Nesterenkonia, Nevskia, Nicoletella, Nitrati ingobium, Sphingomonas, Sphingopyxis, Spirilliplanes, Spir fractor, Nitratireductor, Nitratiruptor, Nitrobacter, Nocardia, illospora, Spirillum, Spirochaeta, Spirosoma, Sporacetige Nocardioides, Nocardiopsis, Nonomuraea, Novosphingo nium, Sporanaerobacter, Sporichthya, Sporobacter, bium, Obesumbacterium, Oceanibulbus, Oceanicaulis, 65 Sporobacterium, Sporocytophaga, Sporohalobacter, Sporn Oceanicola, Oceanimonas, Oceanithermus, Oceanohacillus, lactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Oceanohacier, Oceanomonas, Oceanospirillum, Ochrobac Stackehrandtia, Staleya, Stanierella, Staphylococcus, Stap US 8,785,499 B2 13 14 pia, Starkeya, Stella, Stenotrophomonas, Sterolibacterium, even further other specific aspects, the bacterial NadD is Stigmatella, Stomatococcus, Streptacidiphilus, Streptimono Bacillus anthracis NadD (baNadD). spora, Streptoallomorpha, Streptoalloteichus, Streptobacil In certain aspects of the invention, an antibacterial com lus, Streptobacterium, Streptococcus, Streptomonospora, pound of the invention can be used to treat an infection asso Streptomyces, Streptomycoides, Streptosporangium, Strep ciated with an infectious or toxic biological warfare agent, toverticillium, Subdoligranulum, Subtercola, Succiniclasti including for example, anthrax (Bacillus anthracis), botulism cum, Succinimonas, Succinispira, Succinivibrio, Sulfito (including, for example, Clostridium botulinum toxin types A bacter, Sulfobacillus, Sulfitricurvum, Sulfurihydrogenibium, through G), Brucella species (brucellosis), Burkholderia Sulfurimonas, Sulfurospirillum, Sutterella, Suttonella, Syn mallei (glanders), Burkholderia pseudomalilei (melioidosis), trophobacter, Syntrophobotulus, Syntrophococcus, Syntroph 10 Chlamydia psittaci (psittacosis), Cholera (Vibrio cholerae), Omonas, Syntrophosphora, Syntrophothermus, Syntrophus, Clostridium perfingens (Epsilon toxin), Coxiella burnetii (Q Tatlockia, Tatumella, Taxeohacter, Taylorella, Teichococcus, fever), Cryptosporidium parvum, E. coli O157:H7 (Escheri Telluria, Tenacibaculum, Tepidibacier, Tepidimicrobium, chia coli), epsilon toxin of Clostridium perfingens, a food Tepidimonas, Tepidiphilus, Terasakiella, Terrabacter, Terra safety threat (including, for example, Salmonella species, coccus, Terrimonas, Tessaracoccus, Tetragenococcus, Tet 15 Escherichia coli O157:H7, and Shigella), Francisella tula rasphaera, Tetrathiobacter, Thalassobacillus, Thalasso rensis (tularemia), Lassa fever, Ricintoxin from Ricinus com bacter, ThalassObius, Thalassolituus, Thalassomonas, munis (castor beans), Rickettsia prowaZeki (typhus fever), Thauera, Thaxtera, Thermacetogenium, Thermaerobacter, Salmonella species (salmonellosis), Salmonella Tiphi (ty Thermanaeromonas, Thermanaerovibrio, Thermicanus, phoid fever), Shigella (shigellosis), Staphylococcal entero Thermincola, Thermithiobacillus, Thermoactinomyces, toxin B. Toxic syndrome, a water safety threat (including, for Thermoanaerobacter; Thermoanaerobacterium, Thermoa example, Vibrio cholerae, Cryptosporidium parvum), and naerobium, Thermoanaerolinea, Thermobacterium, Thermo Yersinia pestis (plague)). bacteroides, Thermobifida, Themobispora, Thermo In certain aspects of the invention, an antibacterial com brachium, Thermochromatium, Thermocrinis, pound that selectively binds to an enzyme of the NAD bio Thermocrispum, Thermodesulfatator. Thermodesulfobacte 25 genesis pathway is a compound described in class 1 com rium, Thermodesulfobium, Thermodesulforhabdus, Ther pounds, 3 compounds, and 15 compounds (for example, modesulfo vibrio, Thermoilavimicrobium, Thermohydroge compounds described in Tables 1, 2 and 8-12). In specific nium, Thermonicrobium, Thermomonas, aspects, a compound described in class 1 compounds, 3 Thermomonospora, Thermonema, Thermonospora, Ther compounds, and 15 compounds inhibit a function of NadD. mopolyspora, Thermosediminibacter; Thermosiculum, Ther 30 In other specific aspects, a compound described in class 1 mosinus, Thermosipho, Thermosyntropha, Thermoterrabac compounds, 3 compounds, and 15 compounds selectively terium, Thermotoga, Thermo venabulum, Thermovibrio, bind bacterial Nad) over its human counterpart (e.g., hsNM Thermus, Thetysia, Thialkalimicrobium, Thialkali vibrio, NAT). In further other specific aspects, the bacterial Nad) is Thioalkalimicrobium, Thioalkali vibrio, Thiobaca, Thiobacil Escherichia coli NadD (ecNadD) or Bacillus anthracis NadD lus, Thiobacter. Thiocapsa, Thiococcus, Thiocystis, Thiodic 35 (baNadD). In yet further other specific aspects, the bacterial tyon, Thiohalocapsa, Thiolamprovum, Thiomicrospira, Thi NadD is Escherichia coli NadD (ecNadD). In yet even further Omonas, Thiopedia, Thioreductor. ThiorhodocCocus, other specific aspects, the bacterial NadD is Bacillus anthra Thiorhodococcus, Thiorhodovibrio, Thiosphaera, Thiothrix, cis NadD (baNadD). In specific aspects, class 1 compounds, Tindallia, Tissierella, Toltimonas, Trabulsiella, Treponema, 3 compounds, and 15 compounds inhibit bacterial growth Trichococcus, Trichotomospora, Truepera, Tsukamurella, 40 (for example, by bacterostatic means or bacteriocidal means). Turicella, Turicibacter, unclassified, Ureibacillus, Urubu In certain aspects of the invention, an antibacterial com ruella, Vagococcus, Varihaculum, Vario vorax, Veillonella, pound that selectively binds to an enzyme of the NAD bio Verrucomicrohium, Verrucosispora, Vibrio, Victivallis, Virgi genesis pathway thereby inhibiting its function is adminis bacillus, VirgispOrangium, Vitreoscilla, Vogesella, Volca tered in combination with one or more other antibacterial niella, Volucribacter, Vulcanibacillus, Vulcanithermus, Waks 45 compound. The one or more other antibacterial compound mania, Wautersia, Weeksella, Weissella, Williamsia, can be, for example, an antibacterial compound from a class Wolinella, Woodsholea, Xanthobacter, Xanthomonas, Xeno of antibacterial compounds, including for example, a 2.4- philus, Xenorhabdus, Xvlanibacterium, Xylanimicrobium, diaminopyrimidine, an aminoglycoside, an amphenicol, an Xylanimonas, Xylella, Xylophilus, Yania, Yersinia, Yokenella, ansamycin, a beta-lactam, a carbapenem, a cephalosporin, a Zavarzinia, Zimmermannella, Zobellia, Zoogloea, 50 fluoroquinolone, a glycylcycline, a lincosamide, a macrollide, Zooshikella, Zymobacter, Zymobacterium, Zymomonas, and a monobactam, a nitrofuran, an oxazolidinone, a penicillin, a Zymophilus. polypeptide, a quinolone or quinoline analog, a Sulfonamide, In certain aspects of the invention, an antibacterial com a Sulfone, a tetracycline, or other miscellaneous class of anti pound selectively binds to an enzyme of the NAD biogenesis bacterial compound. In specific aspects, the one or more other pathway thereby inhibiting its function. All three enzymes of 55 antibacterial compound can be, for example, amdinocillin this pathway—NaMN adenylyltransferase (EC 2.7.7.18), (mecillinam), amikacin, amoxicillin, amoxicillin+clavulan NAD synthetase (EC 6.3.1.5) and NAD kinase (EC 2.7.1.23) ate, amplicillin, amplicillin+Sulbactam, atovaquone, azithro (encoded by the conserved genes nadD, nadE and nadF. mycin, aztreonam, bacampicillin, bacitracin, capreomycin, respectively), represent promising broad-spectrum antibacte carbenicillin indanyl Sodium, cefaclor, cefadroxil, cefaman rial targets. In specific aspects of the invention, an antibacte 60 dole, cefazolin, cefdinir, cefditoren, cefepime, cefixime, rial compound selectively binds to and inhibits a function of cefinetazole, cefonicid, cefoperaZone, cefotaxime, cefotetan, NadD. In other specific aspects, the compound selectively cefoxitin, cefpodoxime, proxetil, cefprozil, ceftazidime, cef binds a bacterial NadD over its human counterpart (e.g., tibuten, ceftizoxime, ceftriaxone, cefuroxime and hsNMNAT). In further other specific aspects, the bacterial cefuroxime axetil, cephalexin, cephalothin, cephapirin, ceph NadD is Escherichia coli NadD (ecnadD) or Bacillus anthra 65 radine, chloramphenicol, cinoxacin, ciprofloxacin, clarithro cis NadD (baNadD). In yet further other specific aspects, the mycin, clindamycin, cloxacillin, colistimethate, cycloserine, bacterial NadD is Escherichia coli NadD (ecNadD). In yet daptomycin, demeclocycline, dicloxacillin, dirithromycin, US 8,785,499 B2 15 16 doripenem, doxycycline, enoxacin, ertapenem, erythromy alone in the same or in separate containers, depending on, for cin, fosfomycin, gatifloxacin, gemifloxacin, gentamicin, gre example, cross-reactivity or stability, and can also be supplied pafloxacin, imipenem/cilastatin, imiquimod, kanamycin, in solid, liquid, lyophilized, or other applicable form. The levofloxacin, lincomycin, lineZolid, lomefloxacin, loracar container means of the kits will generally include, for bef, mafenide, malathion, meropenem, methenamine hippu example, a vial, test tube, flask, bottle, Syringe or other con rate, methicillin, metronidazole, meZlocillin, minocycline, tainer means, into which a component may be placed, and moxifloxacin, mupirocin, nafcillin, nalidixic acid, neomycin, preferably, suitably aliquoted. Where there is more than one netilmicin, nitrofurantoin, nitrofuraZone, norfloxacin, novo component in the kit, the kit can contain a second, third or biocin, ofloxacin, oxacillin, oxytetracycline, penicillin, pip other additional container into which the additional compo eracillin, piperacillin+taZobactam, podofilox, polymyxin B, 10 quinupristin--dalfopristin, retapamulin, rifapentine, rifaxi nent may be contained. However, various combinations of min, Saturated solution of potassium iodide, sparfloxacin, components may be comprised in one container. A kit of the spectinomycin, streptomycin, Sulfadiazine, Sulfamethox invention will also typically include a means for containing azole, Sulfisoxazole, Sulphur precipitated in petrolatum, the composition, additional agent, or any other reagent con tainer in close confinement for commercial sale. Such con trichloroacetic acid, bichloroacetic acid, teicoplanin, tellithro 15 mycin, terbinafine, tetracycline, ticarcillin, ticarcillin+clavu tainers may include, for example, injection or blow molded lanic acid, tigecycline, tobramycin, trimethoprim, trimetho plastic containers into which the desired vials are retained. prim+Sulfamethoxazole, trovafloxacin, and Vancomycin. When the components of the kit are provided in one and/or Routes of administration for administering an antibacterial more liquid solutions, the liquid solution is an aqueous solu tion, with a sterile aqueous solution being particularly pre compound of the invention or one or more other antibacterial ferred. The compositions may also be formulated into a compound includes, for example, intraarterial administra Syringeable composition. In this case, the container means tion, epicutaneous administration, eye drops, intranasal may itself be a syringe, pipette, and/or other such like appa administration, intragastric administration (e.g., gastric ratus, from which the formulation may be applied to an tube), intracardiac administration, Subcutaneous administra 25 infected area of the body, injected into an animal, and/or even tion, intraosseous infusion, intrathecal administration, trans applied to and/or mixed with the other components of the kit. mucosal administration, epidural administration, insuffla However, in other embodiments the components of the kit tion, oral administration (e.g., buccal or Sublingual may be provided as dried powder(s). When reagents and/or administration), oral ingestion, anal administration, inhala components are provided as a dry powder, the powder can be tion administration (e.g., via aerosol), intraperitoneal admin 30 reconstituted by the addition of a suitable solvent. It is envi Sioned that the solvent may also be provided in another con istration, intravenous administration, transdermal adminis tainer means. The container means will generally include a tration, intradermal administration, subdermal vial, test tube, flask, bottle, Syringe and/or other container administration, intramuscular administration, intrauterine means, into which the composition is placed, preferably, Suit administration, vaginal administration, administration into a ably allocated. The kit may also comprise a second container body cavity, Surgical administration (e.g., at the location of a 35 means for containing a sterile, pharmaceutically acceptable site of infection), administration into the lumen or paren buffer and/or other diluent. chyma of an organ, or other topical, enteral, mucosal, A kit of the present invention will also typically include a parenteral administration, or other method or any combina means for containing the vials in close confinement for com tion of the forgoing as would be known to one of ordinary skill 40 mercial sale. Such as, e.g., injection and/or blow-molded plas in the art (see, for example, Remington's Pharmaceutical tic containers into which the desired vials are retained. Irre spective of the number and/or type of containers, the kits of Sciences, 18th Ed. Mack Printing Company, 1990, incorpo the invention may also comprise, and/or be packaged with, an rated herein by reference). In certain aspect of the invention instrument for assisting with the injection/administration drawn to administering antibacterial compound of the inven 45 and/or placement of the composition within the body of an tion and one or more other antibacterial compound, the order animal. Such an instrument may be a syringe, pipette, for in which these compounds are administered may be any order ceps, and/or any such medically approved delivery vehicle. (e.g., sequentially or concurrently) and by any route of Examples of compounds are disclosed below. administration. A Compound of Structural Formula 1A:

O O H H N --- rsry's1N As en 1- N A-4 O R 5 R13 R13 R15 O X? (R)s (R1).

In certain aspects of the invention a kit is captured by the 60 wherein invention. In particular embodiments, the invention is drawn As is selected from the group consisting of cycloalkene, to a kit used for treating a bacterial infection. In specific arylene, heteroarylene and polycyclic fused ring, preferably aspects, the kit comprises one or more antibacterial com pounds of the invention for treating a bacterial infection. benzene, naphthalene and anthracene, The kits may comprise a Suitably aliquoted composition 65 each R is independently selected from the group consisting and/or additional agent composition as may be necessary. The of halogen, hydroxy and alkyl, preferably Cl, Br, I and components of the kit may be packaged in combination or methyl: ortho position-Cl; US 8,785,499 B2 17 each R is independently selected from the group consisting of hydrogen and alkyl, preferably hydrogen and methyl; Formula 1 each Rs is independently selected from the group consisting of a carboxy group and an alkyl group Substituted with a carboxy group, preferably —CH2—COOH; and 5 N N1 S s is an integer from 0 to 5, preferably 1. A compound of structural formula 1B: - first (R)s 10 O wherein HN N Aa is selected from the group consisting of aryl, heteroaryl N N Y N and aralkyl, 2 O ) R13 15 preferably (R)s C HO 1 \,

2O wherein Aa is selected from the group consisting of aryl, heteroaryl and aralkyl, preferably 25 where each R is independently selected from the group consisting of halogen, alkyl, hydroxy, and —O-Ra, where Ra is selected from the group consisting of halogen and alkyl, preferably methyl, and t is an integer from 0 to 5: 30

where each R is independently selected from the group 35 CH3 ( ) consisting of halogen, alkyl, hydroxy, and —O—R where Ra is selected from the group consisting of halogen, aryland alkyl, preferably methyl and t is an integer from 0 to 5,

40

each R is independently selected from the group consisting of halogen and alkyl, preferably Cl, Br, I and methyl: ortho 45 position-Cl; each R are independently selected from the group consist ing of hydrogen and alkyl, preferably hydrogen and methyl; and 50 s is an integer from 0 to 5, preferably 1. each R is independently selected from the group consisting of halogen, hydroxy and alkyl, preferably Cl, Br, I and In one embodiment, A is selected from the group consist methyl: ortho position-Cl; ing of each R are independently selected from the group consist ing of hydrogen and alkyl, preferably hydrogen and methyl; 55 and s is an integer from 0 to 5, preferably 1. A pharmaceutical composition comprising the compound 60 of any one of the compounds of formula 1A, 1 02 01, 1 02 02 and 1B as an active ingredient and a pharmaceuti cally acceptable carrier or excipient. Group 01 Compounds where each R is independently selected from the group A pharmaceutical composition comprising at least one 65 consisting of halogen, alkyl, hydroxy, and —O-Ra, where compound of Formula 1 as an active ingredient and a phar Ra is selected from the group consisting of halogen and maceutically acceptable carrier or excipient: alkyl, preferably methyl, and t is an integer from 0 to 5: US 8,785,499 B2 19 20 -continued CH ( ) CH3

CH3

C - Group 03 Compounds 10 A pharmaceutical composition comprising at least one where each R is independently selected from the group compound of Formula 3 as an active ingredient and a phar consisting of Cl, Br, I and methyl; maceutically acceptable carrier or excipient: R is selected from the group consisting of hydrogen and 15 methyl; Formula 3 and A4 s is an integer from 0 to 3. Group 15 Compounds e A pharmaceutical composition comprising at least one A3 N f \ HN-As compound of Formula 15 as an active ingredient and a phar N maceutically acceptable carrier or excipient: S O

Formula 15 25 where A is selected from the group consisting of hydrogen, alkyl and aryl, As is selected from the group consisting of —CONH2 and / \ S (Rs.) wherein Ar is selected from the group consisting of arylene, aralkylene, heteroarylene and aralkyheteroarylene. 35 In one embodiment, where Aris where each Rs is independently selected from the group con sisting of halogen and alkyl, preferably halogen, and p is an integer from 0 to 5, preferably 1 or 2, V-(R1)n S1 (R2), As is selected from the group consisting of 40 --(o ) O --(N= /) where L is selected from the group consisting of alkylene and Y a direct bond, preferably methylene, ethylene, direct bond, 45 branched, rh ( , each R and R2 are independently selected from the group consisting of where each R, is independently selected from the group con halogen, sisting of halogen and alkyl, preferably para-F, and w is an alkyl, 50 integer from 0 to 5, and —N(R) where each R is independently selected from the group consisting of hydrogen and alkyl, preferably both methyl), —O—R where R is hydrogen or alkyl, preferable methyl, —COOR where R is hydrogen or alkyl, preferable methyl, 55 —S R where is alkyl, preferable methyl, —CO Rs where Rs is alkyl, preferable methyl. In another embodiment, Aris In one embodiment, A is selected from the group consist 60 ing of

O 65 Na2 4. S US 8,785,499 B2 21 22 -continued The compound of formula 1M may be:

O

HN NH -N )N1 2 (R)s C 10 Ho1 \,

and the method may produce the compound of structural formula 1 02 2. 15 The compound structural formula 2M may be produced by, for example, reacting with heating to reflux a compound of Formula 3M with a naphthalene-1-carbaldehyde in the pres ence of a solvent:

Compounds of structural formula 1A can be produced by, Formula 2M for example, reacting with heating to reflux a compound of Formula 1M with a benzene-1,4-dicarbaldehyde in the pres ence of a solvent: 25

HN N Formula 1M N ~ O Sa 2. 2 O OH 30 (R)s O Formula 3M O HN NH2 35 N N Y A. 2 O OH where RM is selected from the group consisting of hydrogen (R)s and O 40 where in formulae 2M and 3M, each R is independently selected from the group consisting of halogen, hydroxy and alkyl and s is an integer from 0 to 5. The solvent may be ethanol. 45 The compound of formula 3M may be compound 8 and the compound produced may be formula 1 02 03: each R is independently selected from the group consisting Compound 8 of halogen, hydroxy and 50 alkyl and s is an integer from 0 to 5.

The solvent may be ethanol. 55 The compound of formula 1M may be:

C O Further, the compound of formula 3M may be produced by: Reacting ethyl bromoacetate 3 with tert-butylcarbazate 2 to 60 form (N'-tert-Butoxycarbonyl-hydrazino)-acetic acid ethyl ester 4: N Reacting (N-tert-Butoxycarbonyl-hydrazino)-acetic acid ethyl ester 4 with succinic anhydride to form 4-(N-tert-Bu toxycarbonyl-N-ethoxycarbonylmethyl-hydrazino)-4-oxo 65 butyric acid 5; and the method may produce the compound of structural Mixing 4-(N-tert-Butoxycarbonyl-N-ethoxycarbonylm formula 1 02 1. ethyl-hydrazino)-4-oxo-butyric acid 5 with O-Benzotriazole US 8,785,499 B2 23 24 N.N.N.N'-tetramethyl-uronium-hexafluoro-phosphate CHARMM22 all-atom protein force field and the TIP3P (HBTU) and N,N-Diisopropylethylamine (DIPEA) in N.N- water model S4. In the case of the crystallographic struc dimethylformamide (DMF) and adding an aniline group, tures in 1k4k the sidechain of Trp 117 partially blocks the where said aniline group may be unsubstituted or Substituted targeted binding side. Therefore, the conformation of with at least one group Xselected from halogen, hydroxy and sidechain was searched by performing a two-dimensional X1. alkyl, to form {N'-tert-Butoxycarbonyl-N-3-(X-phenylcar X2 dihedral energy Surface. Following reading of the struc bamoyl)-propionyl-hydrazino-acetic acid ethyl ester 6': tures of monomers A, C and D from 1k4k into CHARMM and Dissolving N-tert-Butoxycarbonyl-N-3-(X-phenylcar adding hydrogens via the IC utility, the energy Surfaces were bamoyl)-propionyl-hydrazino-acetic acid ethyl ester 6' in performed by constraining the remainder of the protein struc Trifluoroacetic acid (TFA) in dichloromethane to form {N- 10 ture and systematically sampling X1 and X2 in 15 degree 3-(X-phenylcarbamoyl)-propionyl-hydrazino-acetic acid increments with an energy minimization to an RMS gradient ethyl ester 7"; and <10 kcal/mol/A at each step in the surface. From the result Dissolving {N-3-(X-phenylcarbamoyl)-propionyl-hy ing energy Surfaces the lowest energy conformation of the drazino-acetic acid ethyl ester 7" in ethanol followed by residue was obtained and used for docking. For all three addition of 1N NaOH to form {N-3-(X-phenylcarbamoyl)- 15 monomers the resulting conformation was such that the propionyl-hydrazino-acetic acid 8'. Trp 117 sidechain did not block the binding site. The resulting Preferably, the aniline group is a 2-chloroaniline; conformation of monomer A of 1k4k was used for the primary compound 6' is butoxycarbonyl-N-3-(2-chloro-phenyl screen of ~ 1 million compounds with those for monomers A, carbamoyl)-propionyl-hydrazino-acetic acid ethyl C and D used in secondary screen one. Additional conforma ester 6: tions of the protein for use in secondary screen two was compound 7" is {N-3-(2-Chloro-phenylcarbamoyl)-pro generated by MD simulation. System preparation for the pionyl-hydrazino-acetic acid ethyl ester 7; and simulation involved obtaining the A monomer of 1k4k, build compound 8' is N'-tert-Butoxycarbonyl-N-3-(X-phenyl ing hydrogens based on the IC facility in CHARMM followed carbamoyl)-propionyl-hydrazino-acetic acid ethyl by a 500 step Steepest Descent energy minimization with the ester 6 in ethanol followed by addition of 1N NaOH to 25 protein non-hydrogen atoms harmonically restrained with a form mass weighted force constant of 1. The system was then {N-3-(X-phenylcarbamoyl)-propionyl-hydrazino-ace overlaid with a preequilibrated box of water designed to be a tic acid 8. minimum of 8 A larger than the protein in the X, Y and Z directions. Water molecules with the oxygenatom within 2.5 III. Examples 30 A of any protein non-hydrogen atom were deleted. The sys tem was then minimized for 500 SD steps with the protein An overview of the structure-based approach applied in harmonically restrained, as above, followed by an additional this study for NadD inhibitor discovery is summarized in 500 step SD minimization of the entire system. The MD FIG. 1. In silico screening of the large virtual library of simulation was initiated from the minimized structure using small-molecule compounds to identify potential NadD 35 the Leapfrog integrator in the isothermic, isobaric (NPT) inhibitors was performed using the ecNadD structural tem ensemble S5 with an integration timestep of 2 fs and plate. Of the ~500 top-ranking in silico hits, 307 commer SHAKE S6 of all covalent bonds involving hydrogens. cially available compounds were Subjected to invitro primary Nonbond interactions were truncated at 12 A with smoothing testing for inhibition of two representative target enzymes, of the Lennard Jones interactions performed via a Switching ecNadD and baNadD. A series of analogs of three high 40 function over 10 to 12 A and the electrostatic interactions ranking compounds of distinct chemotypes (1 ., 3 , and 15 ) Smoothed via a shifting function. The trajectory was contin active against both target enzymes were characterized in ued for 10 ns with the initial 1 ns treated as equilibration, more detail by both enzymatic and cell-based assays. A co coordinate sets were saved every 100 ps. To identify unique crystal structure of baNadD in complex with one of the inhibi conformations of the protein for docking, structures from the tors, 3 02, revealed atomic details of its interactions with the 45 simulations were separated into structurally similar clusters enzyme active site, providing guidelines for future structure using the program NMRCLUST IS7. From this process rep based inhibitor optimization. resentative conformations were obtained from the five largest clusters. These included time frames from 2.1, 5.4, 6.6, 8.5 Example 1 and 9.1 ns. 50 System Preparation for In Silico Database Screening Example 2 The substrate binding site of ecNadDIS1 was selected as Chemical Similarity and Compounds Clustering the target for docking. Visual inspection of the binding region, Solvent accessibility calculations along with consideration of 55 Chemical similarity was determined using the MACCS Bit sequence conservation led to the selection of residues Phe8. fingerprints in combination with the Tanimoto index to define His 19, Ile105 and Ile106 to define the putative inhibitor bind the level of chemical similarity between two compounds S8, ing site. In addition, the level of sequence conservation S9. This procedure allows for all the compounds to be sorted between the bacterial and human enzymes in this region is into clusters where the compounds in each cluster have simi low, thereby maximizing the potential that inhibitors specific 60 lar chemical features S10. One or two compounds were then for bacterial NadD are identified. The apo ecnadD structure selected from each cluster, with the selection being based on (pdb 1k4k) was used for the primary Screen as it represents a physical properties related to Lipinksi's rule of 5 S11, S12. more open form of the binding pocket compared to the prod Application of these rules during compound selection maxi uct deamido-NAD bound form. Molecular modeling and mizes the potential that the selected compounds will have dynamics calculations were undertaken to prepare the protein 65 appropriate bioavailability properties. However, in cases structures for Screening. All modeling calculations were per where clusters did not contain compounds that had all the formed with the program CHARMM S2, S3 using the desired physical properties, compounds were still selected for US 8,785,499 B2 25 26 assay. Chemical clustering and estimation of physical prop were overexpressed in E. coil and purified, and their steady erties was performed using the program MOE (Chemical state kinetic parameters were obtained using a standard Computing Group, Inc.). coupled assay 28. An extensive kinetic analysis of baNadD enzyme, which included detection and exploration of nega Example 3 tive cooperativity, was recently published 24. The results of our previously reported kinetic analysis of this enzyme, albeit In Silico Screening of the Compound Library less detailed, yielded comparable steady state parameters that reflect strong preference for NaMN over NMN 9). A similar The substrate binding site of ecNadD 19 was selected as preference was observed for ecnadD. The experimental test the target for docking. System preparation involved analysis 10 ing of selected compounds for their ability to inhibit NaM of the target protein structure, selection of inhibitor binding NATase activity of NadD enzymes was performed in the site, and generation of the sphere set used to direct the dock 96-well microtiter plate format using a colorimetric end-point ing. The design of the template for in silico Screening was assay, which includes an enzymatic conversion of the based on the 3D structure of ecNadD reported in our earlier releasedPPito Pianda chromogenic reaction with the ammo study 19. The targeted binding pocket encompassed the 15 nium molybdate/Malachite Green reagent 29. nicotinosyl binding site (near residues Asn40, Thr85, Phel04 At this stage of analysis inhibitors with moderate affinity and Ile106 in ecNadD) as well as the catalytic site near the were identified (e.g., ICs at least 100 uM or better). There conserved (H/T)xGH motif (around Phe8, Gly10 and His19). fore, for each of the two enzymes the testing was performed in All database screening calculations were carried out with the presence of compounds at 50-100 uM. The results of DOCK 4.037,38. The primary screening was performed on primary testing of all 307 compounds against both enzymes a 3D database of over 1 million low-molecular-weight com are shown in Table 7. At the 20% inhibition threshold, this mercially available compounds developed in the University method identified 38 ecNadD inhibitors. Remarkably, the of Maryland Computer-Aided Drug Design (CADD) Center baNadD enzyme showed on average a twofold higher Suscep 39, 40. Ligand flexibility was incorporated during docking tibility to inhibition yielding 77 compounds at the same via the anchor-based search method 41. Compounds from 25 threshold. An appreciable correlation across the entire set of the initial primary screen were docked onto the protein based 307 analyzed compounds could be observed in their inhibi on the total ligand-protein interaction energy and scored tory properties against both enzymes (Table 7). This trend can based on the van der Waals (vdW) attractive energies normal be best illustrated by the comparison of two sets of ~10% ized for molecular size 42. top-ranking ecNadD and baNadD inhibitors revealing that Top scoring compounds from the primary screen were 30 nearly one-third of them are shared between both sets (the Subjected to more rigorous secondary docking, where addi estimated probability to get at least 12 random matches is tional optimization of the ligand was performed during the 3x10'). This observation indicated that the applied in silico build-up procedure. Additionally, conformational flexibility screening strategy was indeed Successful in targeting NadD of ecNadD was taken into account via the inclusion of mul active-site components conserved between quite divergent tiple protein conformations either from the crystallographic 35 representatives of this enzyme family. Combining this strat studies (secondary screen A) or from a molecular dynamics egy with the parallel experimental testing of compounds (MD) simulation of ecNadD (secondary screen B). In sec against two divergent target enzymes allowed us to identify ondary screen A, the top 20,000 scoring compounds from the 12 potentially broad-spectrum NadD inhibitors. primary screening were individually docked to the three con formations of apo ecNadD obtained from the 1k4k crystal 40 Example 5 structure. In secondary screen B. multiple protein conforma tions were obtained from the MD simulation of apo ecNadD. Selection and Comparative Analysis of NadD The top 50,000 scoring compounds from the primary screen Inhibitor Analogs were then docked against five MD-generated conformations and ranked using the normalized total interaction energy for 45 To validate and further explore the utility of the three each compound. The top scoring compounds from the two selected chemotypes, structurally similar and commercially separate secondary screens, totaling 500 and 1000, respec available analogs of compounds 1 , 3 , and 15 were iden tively, were then separately Subjected to the final compound tified using chemical fingerprint-based similarity analysis selection based on physical properties and chemical similar 30, 31. For each of the primary compounds, 15 to 40 analogs ity. Determination of chemical similarity and further selec 50 were purchased and analyzed by the same inhibitory assay. tion of compounds were performed according to standard Inhibitory activity above a 20% threshold against at least one procedures. Finally, a total of 529 unique compounds were of the analyzed NadD enzymes was confirmed for 66 of the 89 selected; of these, 307 were purchased from the commercial analogs (Table 8). For example, of the 29 analogs of com Vendors for the in vitro inhibition assay. After primary testing, pound 3 , 23 were active against ecnadD and 24 against three chemotypes (classes 1 , 3, and 15 ) were selected for 55 baNadD, whereas all 18 analogs of compound 1 turned out further analysis of chemical analogs. A total of 89 analogs to be inhibitors of both enzymes. Notably, among 42 analogs were purchased and experimentally tested. of compound 15 , 23 compounds were confirmed as baNadD inhibitors, but only 2 compounds had an appreciable Example 4 inhibitory effect on ecnadD. 60 Overall, an observed frequent occurrence of analogs of Testing of Selected Compounds compounds 1 and 3 that are active against both divergent members of NadD family supports the possibility of devel To evaluate compounds obtained from virtual screening oping broad-spectrum NadD inhibitors. Although all the ana the inventors experimentally tested their inhibitory activity lyzed analogs were selected based only on structural similar against two representative NadD target enzymes, from the 65 ity (without any attempts of their rational improvement), model gram-negative bacterium E. coli and from the Gram many of them displayed a moderate improvement of inhibi positive pathogen B. anthracis. Both recombinant enzymes tory properties compared to the original compounds. For US 8,785,499 B2 27 28 example, 10 analogs of compounds 1 and 3 had improved Each reaction contained 2.3 nM ecNadD (or 1.2 nM activity against ecnadD and 22 against baNadD, pointing to baNadD) in 100 mM Hepes, pH 7.5 buffer, 0.2 mM ATP, 0.07 the possibility of their further optimization. ICs values or 0.2 mM NaMN, 10 mM MgCl, 0.1 mg/ml bovine serum against ecNadD and baNadD determined for a subset of 33 albumin, 0.2 Uinorganic pyrophosphatase, and 50 or 100LM compounds representing all three chemotypes ranged from tested compound (the complete lists of tested compounds low micromolar to >200 micromolar (Table 8). Comparative with structure and vendor information is provided in Tables 1 analysis of these data revealed an appreciable correlation and 2). Bovine serum albumin was included in the assay to (r–0.79) of the inhibitory properties of these compounds reduce the effects of promiscuous inhibitors. against both target enzymes over the entire subset (FIG. 2). The strongest correlation was observed for the compounds 10 The choice of two-fold K, concentrations of both from the most active class 1 (r–0.98). This observation fur NaMN and ATP Substrates was necessary to ensure a good ther confirms the feasibility of developing broad-spectrum signal/noise ratio under the initial Velocity phase of enzy Nad) inhibitors. matic reactions (10-20% substrate depletion), while retaining To assess potential selectivity of these inhibitors against a linear signal response (0-15uMPPi). The same assay setup bacterial targets, several of the most active representatives of 15 was applied when testing Small—molecule inhibitors against each chemotype were tested for their ability to inhibit human human countertargets. Concentrations of hsNMNAT-1 and countertarget enzymes (hsNMNAT-1-3). These assays were hsNMNAT-2 were 3 nM, whereas hsNMNAT-3 was tested at performed at 100LM concentration of the compounds, i.e., in 15 nM. After preincubation of the enzyme with the com the conditions leading to >90% inhibition of bacterial NadD pounds for 5 min at room temperature, the reaction was enzymes. Remarkably, none of the tested compounds dis initiated by addition of NaMN substrate. The reaction was played any appreciable inhibitory activity against the three allowed to progress for 20 min at room temperature prior to human isozymes (<5% for hsNMNAT-1 and hsNMNAT-3, quenching by addition of 100 uL of Malachite Green Reagent and <10% for hsNMNAT-2). These compounds displayed the in 1.2 M sulfuric acid prepared as described by Cogan et al. same efficacy and specificity when tested at a higher concen 29. After 20-30 min incubation to allow for complex/color tration of BSA (1 mg/ml) in the assay, which is a common test 25 formation, the absorbance in each well was measured at 620 to eliminate promiscuous inhibitors 32, 33. Overall, the nm using a microplate reader (Beckman DTX-880). To observed antibacterial selectivity and versatility of the ana account for contribution of free Pi and/or PPi (present in the lyzed inhibitors further support NadD as a promising target sample or released due to nonspecific hydrolysis of ATP for the use and development of broad-spectrum antibiotics. during incubation) as well as of background absorbance 30 (color) of the tested compounds, parallel reactions were run Example 6 for each experimental point without addition of Nad) enzymes, and their W. values were subtracted from the mea Kinetic Analysis of NadD and Primary Testing of Surements of enzyme activity in their respective samples. Selected Compounds Reaction in the presence of 2% DMSO but without inhibitory 35 compound served as a positive control. Each measurement A discontinuous assay was utilized to determine the was made in triplicate. Based on the sensitivity and reproduc steady-state kinetics parametersk, and K for NadD and for ibility of the assay, inhibition >20% was considered reliable. inhibitory testing of selected compounds. This assay couples A continuous coupled assay that detected reduction of pyrophosphate (PP) byproduct formation of NaMNATase NAD28 was used for preliminary assessment of NaMN activity to colorimetric detection of free phosphate released 40 Tase activity and to corroborate kinetic parameters obtained upon enzymatic hydrolysis. with Malachite Green discontinuous assay. (1) NaMN Adenylyltransferase (NaMNATase) Reaction Example 7

(2) Inorganic pyrophosphatase (IPase) reaction 45 ICs. Measurements and K, Determination The compounds selected based on the results of primary Excess IPase is used to ensure rapid conversion of pyro testing were further characterized using the malachite green phosphate to orthophosphate so that the rate-limiting step in end-point assay. The initial rate of enzymatic reaction was this system is the NaMNadenylyltransferase reaction. Excess 50 measured at fixed NaMN and ATP concentrations (equal to inorganic phosphate also decreases the probability that two-fold K values) and various concentrations of an inhibi observed inhibition is due to the inhibition of IPase and not tory compound. The ICso value was determined by plotting the relative NaMNATase activity versus inhibitor concentra the target enzyme. The inventors confirmed that the best tion and fitting to the equation (1) using GRAPHPAD NadD inhibitors (with ICs values ranging from 5 to 25uM) PRISM. did not inhibit IPase. 55 Steady-state kinetic analysis of ecNadD and baNadD tar get enzymes was performed by varying substrate (NaMN or ATP) concentrations were 0, 10,30, 60,200, 500 uMat fixed (1) saturating concentration of second Substrate (0.5 mM). Cso Apparent values of K, and k were calculated by fitting 60 initial rates to a standard Michaelis-Menten model using the Software GRAPHPAD PRISM. Vo and V, represent initial rates in the absence and presence of The standard inhibition assay was configured in a 96-well inhibitor concentration I. format for automated liquid-handling and convenient read For K, determination, the enzyme was preincubated with out. Each compound was prepared as a 10 mM stock Solution 65 various fixed concentrations of inhibitors for 5 min. The in dimethyl sulfoxide (DMSO) and diluted tenfold (10% reaction was initiated by the addition offixed concentration of DMSO) before usage. NaMN (five-fold K) at varying concentrations of ATP (rang US 8,785,499 B2 29 30 ing from 0.2 to fivefold K) and vice versa. The inhibition tion of compound that caused more than 50% growth inhibi constant and inhibition pattern were evaluated by fitting the tion (as determined by AUGC method). data to the Michealis-Menten rate equation (2) for general The antibacterial activity of selected NadD inhibitors was (mixed-type) inhibition 43 with the program GRAPHPAD assessed by their ability to suppress the growth of model Gram-negative (E. coli) and Gram-positive (B. subtilis) bac PRISM. teria in liquid culture. To establish conditions potentially maximizing the effect of NadD inhibition in an E. coli model, VaS (2) AnadA mutant strain with disrupted de novo NAD synthesis W = were use. To further restrict the flux of NaMN (the committed K. (1 -- )+(SIC -- I substrate of the NadD target enzyme) growth studies on the i ...) 10 experimentally established lowest concentration of Nam (0.4 uM) Supporting the growth of this diagnostic strain on mini mal media were performed. In these conditions, many of the V and K, are standard Michaelis-Menten parameters, and selected NadD inhibitors of classes 1 and 3 showed an appre K, is the equilibrium dissociation constant for the enzyme ciable growth suppression effect at 100 uM (FIG. 3A and inhibitor complex. The parameter a defines the degree to 15 Table 8). To assess the extent of “on-target” (Nad D-depen which the inhibitor binding affects the affinity of the enzyme dent) versus “off-target' (nonspecific) antibacterial effects of for the substrate and is diagnostic of the inhibition mode, these compounds, an E. coli strain containing an overexpres which may be purely competitive (CD1), purely noncom sion plasmid vector with the E. coli nad D gene was used. The petitive (C=1), uncompetitive (C.<1), or mixed-type (CD1 or growth of this strain in the presence of selected inhibitors was C.<1). compared to an isogenic control strain containing the same plasmid vector overexpressing a housekeeping gap A gene Example 8 (unrelated to NAD synthesis). As shown in FIGS. 3C and 3D, overexpression of ecNadD suppressed the antibacterial activ Suppression of Bacterial Growth in Culture ity of the tested representatives of NadD inhibitors of classes 25 1 and 3. On the other hand, the bactericidal effect of the compound 15 11 (Table 2) was essentially the same in both E. coli strains used for growth-Suppression experiments the NadD-overexpressing and control strain suggesting that and for target verification were prepared in the background of this effect is largely non-specific (NadD-independent). An the E. coli K-12 BW25113 (AnadA) knockout strain with alternative interpretation that the on-target activity of 15 11 disrupted NAD de novo synthesis pathway from the Keio is too high to be suppressed by NadD overexpression appears collection (a gift by Dr. H. Mori, Keio University, Japan)44. 30 unlikely, as the invitro inhibitory properties of this compound This strain was used in combination with one of the two are below average (ICson-200LLM). Based on the struc expression plasmids from the E. coli ASKA library 45 ture of this compound, one may expect its hydrolysis in the enabling inducible overexpression of the: (i) E. coli nadD medium to benzoate, a compound known to have a general and non-specific antibacterial activity. gene (to test for the increased resistance against NadD inhibi 35 An appreciable antibacterial activity was also observed for tors) or the (ii) E. coli gap A gene, a housekeeping metabolic several analogs of compounds of class 1 and 3 against the enzyme glyceraldehyde-3-phosphate dehydrogenase (as a model gram-positive bacteria B. subtilis (Table 2 and Table negative control). Starter cultures were grown overnight in 8). Interestingly, the antibacterial effect of tested compounds LB medium. Cells were harvested, washed, and resuspended in B. subtilis was manifested by delayed growth in contrast to in the M9 minimal growth medium containing 1% glycerol, 40 E. coli where it was largely a decreased final cell density (FIG. 0.1 mMIPTG, 50 mg/l kanamicin, 35 mg/l chloramphenicol 3B). Although establishing a rationale for this difference and and a limiting amount of nicotinamide (Nam, 0.4LM). Upon confirming the actual target in Gram-positive bacteria remain reaching an optical density at 600 nm of 0.05, cells were used to be accomplished, the growth-Suppression data shown in to initiate growth experiments in 96-well plate at various Table 2 indicate that Nad) inhibitors do indeed function as concentrations of inhibitors. 45 broad-spectrum antibiotics. MIC for active compounds The bacterial growth at 37°C. in these (and other) experi 3 02, 3 05, 3 15, 3 23, and 1 03 against B. anthracis ments was monitored by continuous absorbance measure sterne, B. subtilis, and E. coli was determined. A general ment at 600 nm using an orbital shaker/microplate reader correlation was observed between Nad) inhibition and anti ELX808TM. The area under the curve (AUGC) was used to bacterial activity, although being less pronounced in E. coli calculate the growth inhibition 46 and was compared to the 50 (Table 2). Cell wall impermeability of gram-negative bacteria respective amount of DMSO. The AUGC was integrated and could be a major determinant of Such weaker Susceptibility. calculated with GRAPHPAD PRISM. Growth suppression Notably, some of the less efficient ecnadD inhibitors (e.g. studies of B. subtilis 168 (Bs168) were performed following 3 23 and 15 11) showed a relatively strong antibacterial a similar procedure in a chemically defined medium 47 activity against E. coli. This observation may reflect the exist containing glucose (4 g/l), tryptophan (50 mg/l), glutamine (2 55 ence of additional targets affected by these compounds, non g/1, KHPO (10 g/l), KHPO (6 g/l), sodium citrate (1 g/l), specific or even sharing some common features with NadD MgSO4 (0.2 g/l), K2SO4 (2 g/l), FeC1 (4 mg/l), MnSO (0.2 34. In addition the inventors demonstrated that 01 02 01 mg/l). B. anthracis was grown in the same minimal medium (RK-AL-1) (see Table 12) are effective ecNadD and baNAD containing additionally 10% LEB medium for robust growth. inhibitors (FIG. 4). Selected compounds causing an appreciable growth inhi 60 bition were subject of minimal inhibitory concentration Example 9 (MIC) determination in a series of dilutions from 160 uM downto 2.5uM. The high concentration limit was determined Mechanistic and Structural Analysis of NadD by solubility problems observed for many compounds. In this Inhibition concentration range only some of the analyzed compounds 65 displayed >90% growth inhibition. Therefore, for consis Representatives of both classes 1 and 3 of efficient NadD tency, the value of MIC was defined as the lowest concentra inhibitors were selected for detailed kinetic characterization US 8,785,499 B2 31 32 and co-crystallization trials. Apparent steady-state inhibitory dimer interface were observed in the crystal structure, it is parameters were obtained for compounds 1 02 and 3 02 unlikely that such interactions would contribute to the inhi against ecNadD and baNadD with respect to each substrate bition observed in our assay conditions. This conclusion is ATP and NaMN (Table 1 and FIG. 18). A preliminary assess based on the fact that the enzyme concentration in the assay ment of all kinetic profiles revealed a mixed-type inhibition as (~1 nM) was substantially lower than the dimer K, (0.11 uM) indicated by CD1 values obtained by fitting initial rates to a as estimated by AUC analysis. Moreover, AUC data did not general inhibition model. Despite the observed complex reveal any changes in the oligomerization state of the protein behavior preventing a straightforward mechanistic interpre in presence of the inhibitor. Therefore, the contribution of the tation, the obtained data showed a substantial similarity in the handshake dimer interface to baNadD inhibition by 3 02 inhibitory properties of both compounds with respect to both 10 should be negligible under the assay conditions. This conclu target enzymes. sion is consistent with the fact that ecNadD, being monomeric The 3D structure of the complex of baNadD co-crystal both in the crystal structure and in solution, exhibits essen lized with compound 3 02 and solved at 2.0-A resolution tially the same inhibitory properties in the presence of 3 02, revealed its binding in the active-site area mostly through Van including the same mixed-type mode and similar kinetic der Waals interactions. The planar compound stacks against 15 parameters. two aromatic residues, Trp116 and Tyr112 (baNadD number Notably, the three most flexible regions in baNadD men ing), and is also in contact with Met 109 and Phe 103 (FIG. tioned above also correspond to the regions that deviate the 6A). While there are a few water-mediated indirect interac most from the hsNMNAT structure 15 (FIG. 19). Compari tions between 3 02 and the enzyme, there is no direct inter son of human NMNAT structures (as represented by hisNM molecular hydrogen-bond interaction. A comparison with the NAT-115) with various baNadD complexes indicated that 3D structure of baNadD complexed with the NaAD product hsNMNAT active site conformation is much closer to the solved in this study at 2.2-A resolution and with the recently product-bound conformation of baNadD than to the apo form reported apo-baNadD structures 21, 24 provided additional of baNadD. No significant conformational change has been insights to the structural mechanism of inhibition. This com observed between the apo and ligand bound human NMNAT parison revealed that the bound compound 3 02 partially 25 enzymes 15, 35, 36. Therefore the active site of human overlaps with the nicotinosyl binding site and would interfere NMNAT, being quite dissimilar from the apo or inhibitor with NaMN substrate binding (FIG. 6B). In particular, inhibi bound baNadD, appears unable to accommodate or specifi torbinding would potentially block the critical stacking inter cally interact with inhibitor 3 02 (FIG. 19). This interpreta action between the side-chain of the conserved Trp 116 resi tion is Supported by the results of comparative virtual docking due with the pyridine ring of the NaMN substrate 19, 22. 30 performed for the three classes of active compounds against This interference may contribute to a competitive aspect of ecNadD, baNadD, and hsNMNAT1. The docking energies the observed mixed-type inhibition. for the human enzyme consistently have the least favorable The structure comparison also revealed a substantial dif scores compared to the energies obtained for ecNadD and ference between the active-site conformations in the baNadD, especially in the van der Waals energy terms, Sug baNadD-3 02 and baNadD-NaAD complexes. Moreover, 35 gesting that the overall shape of the binding region in hisNM the active-site conformation in the baNadD-3 02 complex is NAT is sufficiently different to allow for selective inhibition much more similar to apo-baNadD (rmsd between C atoms of bacterial enzymes. 0.77 A) than to the baNadD-NaAD complex (rmsdof 1.32A). The major conformational differences occur in the regions Example 10 that are involved in NaMN binding, i.e., residues 42-48 (loop 40 connecting B2 and O2), 105-126 (helix C4), and the loop Computational Analysis of Inhibitor Selectivity between B5 and B6 (residues 131-149) (FIG. 6B). Notably, these flexible regions correspond to the three regions that Docking of selected compounds (Table 2) was performed deviate the most from the hsNMNAT structure 15. Without targeting the binding region into which compound 3 02 was being bound by theory, in addition to interfering with NaMN 45 observed to bind in the crystal structure. Docking targeted all substrate binding, the interactions between the inhibitor and available crystal structures of the E. coli (n=2), B. anthracis baNadD may partially “lock” the enzyme active site in the (n-6) and human (n=4) forms of the enzyme (Table 3). catalytically impaired apo-like conformation. This mecha Sphere sets to direct docking were generated using the SPH nism provides a rationale for the observed, largely noncom GEN, selecting sphere sets located in the binding region petitive mode of inhibition described above. 50 defined by 3 02 in the crystal structures. Residues adjacent The baNadD enzyme has a tendency to form a homodimer to the sphere sets are listed in Table 3. Docking was per as observed in the crystal structure of both, apo-form and of formed for each compound against each crystal structure its complex with Substrate and confirmed by size-exclusion using the secondary Screening approach. Table 4 includes the chromatography and analytical ultracentrifugation (AUC) most favorable Dock energy scores for each compound over (data not shown). Inspection of baNadD-3 02 complex crys 55 all the crystal structures for each the three species. With tal packing shows that while the native dimer interface is respect to the E. coli and B. anthracis enzymes there is no preserved, an additional dimer interface, similar to that of the appreciable correlation with the ICs values reported in Table “handshake' dimer observed in B. subtilis NadD 22 is also 2. For example, the ICso values of 1 02 are similar, while present, resulting in a tetrameric appearance. The 3 02 docking predicts binding to ecNadD to be favored while with inhibitor binding site is located at this hand-shake dimer 60 3 23 the more favorable energy with the baNadD is consis interface. Because the compound binds at a symmetrical site tent with the relative ICs values. An interesting outcome of between two baNadD monomers related by a pseudo-twofold the docking is that a larger number of the compounds have symmetry, the two symmetrical orientations of 3 02 cannot docking energies that are more favorable with baNadD then be distinguished. Therefore, 3 02 was modeled in both ori with ecnadD (9 versus 6, respectively). This may indicate entations, each with half occupancy. 65 that while docking was performed targeting ecnadD, there is Although additional interactions between the compound some inherent property of baNadD that leads to favorable 3 02 and the adjacent baNadD subunit at the handshake ligand-protein interactions. However, this result may be due US 8,785,499 B2 33 34 to the docking analysis being performed against 6 conforma ecules in the crystal. Therefore Applicants modeled 1 02 tions of baNadD versus 2 for ecNadD, where the larger num molecule in two orientations each with half occupancy (FIG. ber of conformations increases the probability that a confor 8). mation more Suitable for a given ligand is targeted. Perhaps baNadD structures have been reported recently in its apo more significant are the results when the docking energies are form, in complex with substrate NaMN, with product NaAD, compared for all three species. For all but two of the 15 as well as with inhibitor 3 02''' ' '. The overall compounds the least favorable score occurs with hsNMNAT, baNadD structure contains a Rossman-fold core with a cen with the most favorable score occurring for only one com tral six-stranded parallel B-sheet and two or three C. helices on pound (3 05). To better understand the types of interactions each side of the sheet (FIG.9A). Following the sixth and the leading to the less favorable scores with hisNMNAT, the elec 10 last 3 strand, two a helices (C.6 and C.7) form a small C-ter trostatic and VdW ligand-protein interaction energies were minal subdomain that is characteristic of the nucleotidyl examined. Results in Table 5 show the most favorable elec transferase superfamily. The signature HXGH motif trostatic interaction energies to often occur with the human (HYGHs) is located in the loop connecting the first enzyme while the most unfavorable most often occur with B-strand (B1) and Succeeding C. helix (C.1). This motif is ecNadD. In contrast with the vdW energy, in the majority of 15 involved in the interaction with the phosphate groups of the cases the human enzyme term is the least favorable, with only substrates (ATP and NaMN) and participates in the catalysis. two exceptions. To more closely examine the nature of the In the baNadD-1 02 complex structure, 1 02 sits at a vdW contribution the attractive vaW interaction energy was central cleft between strands B1 and B4 of the B sheet, which calculated. The attractive vaW interaction represents the is the catalytic and substrate binding sites of the enzyme (FIG. quality of the steric fit of a ligand with the protein, such that 9A). The compound is bent at the acylhydrazone linkage and it is used as the compound scoring criteria for the primary follows the contour of the crevice of the substrate binding site screen methodology in this study. Results in Table 6 show that (FIGS. 9B and 9C). The anthryl rings together with the acyl hsNMNAT has the least favorable attractive vaWinteraction hydrazone of the compound stack against the side chains of energy in all but one case. Although the docking approaches Trp 116, Tyr112 and Met109 (FIG.9B). A single direct hydro and, scoring functions used in the analysis are very approxi 25 genbond is formed between the amide group of the carboxya mate, the observed vdW terms were consistently most unfa mide moiety of 1 02 to the main chain carbonyl of Gly8. The vorable for hsNMNATsuggesting that the overall shape of the chloride of the terminal chlorophenyl group appears to inter binding region of the human enzyme differs enough from that act favorably with the side chain of His 18 of the HXGH motif. of ecNadD and baNadD to afford the observed selective inhi There are two indirect hydrogen bonds between the com bition. The attractive vaW results show baNadD to typically 30 pound and protein atoms. One is formed between the hydra have the most favorable values. Zone amide and the side chain of Thr85 via a water molecule Additional Results (watt), and the other between the acyl oxygen group and Structure of baNadD in Complex with Inhibitor 1 02 Asn39 side chain through wat1. The chlorophenyl ring is also The complex of baNadD and 1 02 crystallized in the same in contact with the side chains of Ile 7 and Ile 21, which may space group P222 as the previously reported baNadD-3 02 35 provide additional stabilizing van der Waals interactions with complex'' and the protein conformations in the two inhibi the compound (FIG.9B). tor complexes are also very similar with root mean square Comparison of the Binding Modes of 1 02 and 3 02 deviation (RMSD) for all Catoms of 0.175A; they resemble Comparison of the binding mode of 1 02 and that of 3 02 the conformation of the enzyme in its apo state rather than the reported previously' shows that the nearly coplanar substrate or product bound state, with RMSD values of 0.494 40 anthracene rings and the hydrazone portion of 1 02 overlaps A and 0.833A, respectively, compared to the apo and product with the largely planar 3 02 (FIG. 10A). They form similar bound baNadD''': ' ' (Reference A14, Sorcietal, Tar stacking and Vander Waals interactions with multiple protein geting NAD biosynthesis in bacterial pathogens. Structure residues including Trp 116, Tyr112, Met109 and Lys115. This based development of inhibitors of nicotinate mononucle shared binding site corresponds to the region that binds nico otide adenylyltransferase NadD, Chem Biol, Aug. 28, 2009, 45 tinic acid riboside portion of NaMN substrate in the absence 16, 849-861 and Sorci et al. Supplemental Data. Targeting of the inhibitors (FIG.10B). In particular, Trp 116 would stack NAD biosynthesis in bacterial pathogens: Structure-based against the pyridine ring of NaMN and is critical for the development of inhibitors of nicotinate mononucleotide ade proper positioning of the substrate. Therefore binding of the nylyltransferase NadD, Chem Biol, Aug. 28, 2009, 16, 849 inhibitors would prevent NaMN binding all together. Nota 861 and Huang et al. Complexes of Bacterial Nicotinate 50 bly, the two classes of compounds do not overlap completely Mononucleotide Adenylyltransferase with Inhibitors. Impli and each has additional interactions with the enzyme that are cation for Structure-Based Drug Design and Improvement, J. not present in the other compound (FIG. 10A). While 3 02 Med. Chem. Jun. 25, 2010 (web) are each hereby incorpo largely overlaps with the NaMN substrate binding site, 1 02 rated by reference in their entirety. also intrudes into the ATP binding pocket and its chlorophe Inspection of the electron density for the bound compound 55 nyl group would overlap with the ribose of ATP (FIG. 10B). revealed a symmetrically shaped density much larger than the The inhibitory efficiencies of the two compounds against compound (FIG.8A). This density is located at a symmetrical haNadD have been determined previously, with 1 02 having interface between two baNadD monomers where the inhibi K of 9 LM and 10 uM, respectively, with regard to NaMN and tor can bind in one of two different but symmetrically related ATP substrates; while 3 02 has K, of 18 uM and 32 uM orientations, with the positions of the central anthracene ring 60 against NaMN and ATP, respectively'. These values are overlapping with each other (FIGS. 8A and 8B). These two consistent with the structural observation that 1 02 inter orientations are in fact equivalent and physically indistin feres with binding of both NaMN and ATP whereas 3 02 guishable. It can be viewed as Such that in the complex crys mostly interferes with NaMN binding. tal, half of the protein molecule population would bind the Binding of both 1 02 and 3 02 appears to stabilize the inhibitor in one orientation, while the other half bind the 65 enzyme in a conformation that is significantly different from inhibitor in the second orientation. The resulted electron den its substrate or product bound form, and is apparently cata sity is the accumulated average from all the complex mol lytic incompetent (FIG. 10B). The conformational differ US 8,785,499 B2 35 36 ences associated with inhibitor binding as compared to the interactions between the acylhydrazone amide group and the Substrate or product bound conformations has been Suggested main chain amide and side chain hydroxyl of residue Thr85 to lead to mixed inhibition kinetics that contains both com (FIG. 12C). In the NaMN or NaAD complex structure, the petitive and non-competitive characters''. carboxylate group of the nicotinic acid binds in this region Structure of baNadD in Complex with Inhibitor 1 02 1 and interacts with the main chain amide of Thr85. Thus, the Because 1 02 must adopt either of the two symmetrically formate molecule observed in the 1 02 1 complex structure related orientations with half occupancy in the crystal due to mimics the interaction between the nicotinic acid carboxylate the overlapping position of the anthracene rings, Applicants group of the substrate NaMN and the enzyme. hypothesized that a symmetrical compound that fit the Carboxylate Containing Analogs of 1 02 and 1 02 1 observed density of 1 02 would bind to the enzyme with full 10 Motivated by the presence of the formates in the 1 02 1 occupancy and higher affinity. A compound was designed to complex structure, additional analogs were designed. These retain the central planer ring system with an acylhydrazone analogs (1 02 2 and 1 02 3 in Scheme 3) (FIG. 17) were arm and terminal chlorophenyl ring on each side. The result designed to include a carboxylate moiety to approximate the ing compound, designated 1 02 1 (Scheme 1) (FIG. 15), location of the formates in the baNadD-1 02 1 complex was synthesized and Subjected to biochemical and crystallo 15 structure. In that structure, one formate oxygen is 2.76 A from graphic analysis. Compound 1 02 1 replaced the the side chain hydroxyl group of Thr85 and the other oxygen anthracene ring with a benzene and includes a Cl atom at the is 2.91 A from the backbone amide nitrogen of Thr85, form ortho position of the terminal phenyl rings; this selection was ing a well-defined ion-dipole interactions. Accordingly, it based on availability of chemical precursors. 1 02 1 was was hypothesized that the carboxylate moieties would mimic then tested as a NadD inhibitor. According to the structure these interactions, thereby further improving binding. In activity relationship (SAR) data from a limited set of analogs addition, the inclusion of the carboxylate moieties would of Class 1 compounds (FIG. 11A), it was expected that enhance the Solubility of the compounds, making them more 1 02 1 should have an improved activity compared to the Suitable for biochemical experiments and potentially enhance different asymmetric "monomeric' compounds. Inhibition their bioavailability. This led to the design and synthesis of assay on 1 02 1 yielded an ICs of 13+2 uM and 16+4 LM 25 1 02 2 and 1 02 3 shown in Scheme 3 (FIG. 17). against ecNadD and baNadD, respectively (FIG. 11B). Com 1 02 2 was a direct mimic of 1 02 1 while 1 02 3 was pared to the Class I analogs shown in FIG. 11A, 1 02 1 is designed as an analog of 1 02, to test if the presence of the significantly better than those compounds with either a ben carboxylate could improve the affinity of the monomeric Zene or naphthalene rings, while its activity is similar to those species. compounds containing an antharcene ring, including 1 02. 30 Experiments were then undertaken on the two new 1 02 As 1 13 and 1 15 in FIG. 11A contain only benzene rings analogs to measure the inhibitory activity against baNadD. and linkers identical to 1 02 1, they may be considered as Surprisingly, 1 02 2 did not inhibit baNadD at concentra “precursors” of 1 02 1. Therefore the design strategy to tions up to 100 uM, while 1 02 3 only weakly inhibits create a symmetrical compound may be considered Success baNadD activity (ICs >200 uM). Thus, the inclusion of the ful, as a more than 10 fold improvement in activity was 35 carboxylates did not lead to improved binding with the sym achieved. 1 02 1 is also slightly more active than com metric, dimeric analog 1 02 02, although some binding pound 1 02, which has an ICs of 25uM. affinity of the monomer analog, 1 02 3 is present. To understand the binding mode of 1 02 1, Applicants To understand the unexpected results, both 1 02 2 and determined the crystal structure of baNadD in complex with 1 02 3 were Subjected to crystallographic analysis. All the compound. The baNadD-1 02 1 complex has the same 40 attempts to cocrystallize 1 02 2 with baNadD were unsuc crystal form as the 1 02 complex and retains the crystal cessful; however, cocrystals of 1 02 3 bound to baNadD lattice packing involving the same monomer-monomer inter were obtained and the complex structure was determined to face to which the inhibitor binds. 1 02 1 has well defined 2.55 A resolution. The 1 02 3 complex crystal is in a dif electron density and is modeled with full occupancy (FIG. ferent space group (C2) from all other baNadD-inhibitor 12A). As predicted, 1 02 1 binds at the same site as 1 02 45 complexes, and contained eight baNadD monomers in the and overlaps with the two orientations of that molecule (FIG. asymmetric unit. Notably, the baNadD monomer-monomer 12B). The conformations of the acylhydrazone arms of the interface to which 1 02, 3 binds is different from that two compounds are very similar despite the presence of sev observed in all other inhibitor complex structures (FIG. 13A), eral rotatable bonds (FIG. 12B). 1 02 1 also interacts with indicating that packing of the enzyme molecules in the crystal the protein similarly as 1 02. Most van der Waals interac 50 can be influenced by inhibitor binding. The electron density tions, especially the Stacking interactions with Trp116 and for 1 02 3 was well-defined allowing unambiguous mod Tyr112, are preserved (FIG. 12C). However, due to the dif eling of the compound in the complex (FIG. 13B). Interest ference in the central ring systems and the restriction of the ingly, the binding mode of 1 02 3 differs significantly from covalent linkage to the central benzene ring, the acylhydra that of 1 02 and 1 02 1, although some overlap is present Zone arms of 1 02 1 displays a slight rigid body rotation 55 (FIG. 13C). In particular, the naphthalene ring of 1 02 3 (~15 compared to 1 02. As a result, there are differences in binds to the same site as the aromatic rings of the other Class the hydrogen bond patterns and in the orientation of the end 1 compounds, and form similar van der Waals contacts with chlorophenyl group. The hydrogen bond between the car Trp116, Tyr112, Met 109, as well as with Lys115. However, boxyamide nitrogen of 1 02 and Gly8 main chain (shown in the acylhydrazone arm and the end chlorobenzene ring of FIG. 9B) is lost in the 1 02 1 complex structure, whereas a 60 1 02 3 adopt completely different conformations from that new hydrogen bond is formed between the carboxyamide of compounds 1 02 and 1 02 1, and interact with different oxygen and Gly 106 main chain amide (FIG. 12C). The functional groups on the protein. In this binding mode, the Smaller single six-membered central ring of 1 02 1 may carboxylate group of the compound, though occupying a lead to a decrease in the van der Waals interactions with the similar position as the formate molecule in the 1 02 1 com protein as compared to the anthracene ring in 1 02. 65 plex, interacts with the enzyme somewhat differently. One Interestingly, in the 1 02 1 complex structure, two well oxygen of the carboxylate still interacts with the side chain ordered formate molecules are observed mediating specific hydroxyl of Thr85 (3.13 A). In addition, there are ion-dipole US 8,785,499 B2 37 38 interactions of the carboxylate with the main chain amides of structure, binding of the carboxylate group at this site comes Thr85 (3.30 A) and of Tyr117 (2.81 A) (FIG. 13D). These at the expense of completely reorienting the acylhydrazone interactions are reminiscent of those observed in the NaMN arms of the compound which results in an overall decreased substrate complex where the carboxylate of the substrate also affinity. This reorientation is also proposed to disallow bind forms two hydrogen bonds with the main chain amides of 5 ing of the dimeric 1 02 2 to the protein. both Thr85 and Tyr117'''. An additional hydrogen bond Although the activities of the current Nad) inhibitors are between the amide group of 1 02 3 and the main chain only in the low micromolar ICso range at best, there are carbonyl of Lys115 is also observed (2.8 A). Overall, these several attractive features in their binding modes. Binding of interactions lead to a different binding mode for 1 02 3 both classes of inhibitors appears to stabilize the enzyme in a eventhough the aromatic and acidic groups bind to the sites as 10 catalytically incompetent conformation, significantly differ predicted based on the 1 02 1 complex. In this mode the ent from its Substrate or product bound conformation, result carboxy amide moiety and adjacent chlorophenyl ring of the ing in a mixed inhibition kinetics behavior that contains both compound are largely exposed to the solvent while their competitive and non-competitive characters. As such the counterpart in the 1 02 and 1 02 1 complexes binds to the binding pocket can accommodate Small molecules with struc adenosine binding site of the enzyme and is much less solvent 15 tures very different from the natural ligands of the enzymes. accessible. Therefore, such small molecule binders are anticipated to The 1 02 3 complex structure provides a possible expla have minimal adverse effects on the numerous other NAD" or nation as to why 1 02 2 does not bind as anticipated. While ATP utilizing enzymes. The non-competitive character of the naphthalene ring and carboxylate moieties bind to the inhibition by these inhibitors also indicates that once higher anticipated sites, the geometrical restraints to achieve these affinity compounds are found, they may not be strongly influ interactions leads to a reorientation of the compound and a enced by cellular ATP or NAD" concentrations, which are on significant change in the overall binding mode of 1 02 3 the order of ~10-10 uM'''. Such inhibitors could have (FIGS. 13C and 13D). Potential binding of 1 02 2 in the better in vivo efficacy than purely competitive inhibitors. same orientation as 1 02 3 would disallow the second arm Although a non-native dimer interface is observed in sev of the hydrazine linker to access the binding pocket occupied 25 eral baNadD-inhibitor (e.g., 3 02, 1 02 and 1 02 1) com by 1 02 and 1 02 1, thereby abolishing binding. plex crystal structures, it has become clear that this dimeriza Discussion tion mode is due to crystal lattice packing interactions under In an effort to develop inhibitors targeting the essential specific crystallization conditions since Such dimerization is bacterial NadD enzymes, Applicants have identified three not observed in Solution in an analytical ultracentrifugation classes of bacterial NadD inhibitors with distinct scaffolds in 30 study''. Crystal structures of 1 02 3 complex and apo a structure-based in sillico screen''. Applicants have also baNadD obtained in different space groups also do not have obtained the crystals structures of B. anthracis Nad) in com the same dimerization mode' '. This observation par plex with inhibitors from two different chemical classes: tially explains the moderate improvement of the activity of 3 02 from Class 3 (reported in Ref. 14), and three different the much larger dimeric 1 02 1 as compared to its mono Class 1 compounds (1 02, 1 02 1 and 1 02 3). The 35 meric precursor. Therefore future inhibitor design and opti complex structures of baNadD with different inhibitors mization effort should be focused on engineering specific revealed a common binding site near residues Trp116. direct interactions between the inhibitors and enzyme mono Try 112, and Met109, as shown in FIG. 14, which appears to mer. Toward this goal, the complex structures of NadD with have an affinity for aromatic groups from different small different inhibitors provided useful information on a common molecules. This site overlaps but is distinct from the substrate 40 primary target site and the chemical environment of the vicin NaMN binding pocket, and may serve as a primary site to be ity of this site which can be exploited to improve on the targeted in future inhibitor design efforts. Such design efforts existing inhibitor scaffolds or design high affinity inhibitors would target compounds whose aromatic moieties interact with novel scaffolds for maximum interaction with the with the identified “aromatic' site, with the remainder of enzyme. those putative molecules sampling various binding modes in 45 Experimental Section the vicinity of this site. Protein Crystallography The compounds of the present invention also include ones The expression and purification of Bacillus anthracis that interact with an inhibitor binding pocket of baNAdD at NadD (baNadD) has been reported elsewhere'''. For co one or more residues selected from Trp 116, Tyr112, Met 109, crystallization of baNadD with compounds 1 02, 1 02 1 Lys115 and Phe 103. The compounds of the present invention 50 and 1 02 3, appropriate amount of the stock compound are not limited to ones that interact with an inhibitor binding solutions (20 mM in DMSO) was mixed with the protein to pocket of baNAdD, but may also be ones that interact with a the final concentration of 1 mM, while the final protein con similar site defined by homologous residues on any bacterial centration is 19 mg/ml. The PEG/Ion Crystallization Screen NadD protein. FIGS. 6-10, 12-14 and 19 also show some ing kit (Hampton Research) was used for the initial screens of residues that are not located at the common binding site. 55 the complex crystals. Hanging drop vapor diffusion methods The complex structures of three Class 1 compounds pro were used for the crystallization where equal volume (1.5ul) vide useful information about the chemical characters of the of the complex and reservoir Solution was mixed and equili inhibitor-binding site of NadD. Compounds 1 02 and brated against the reservoir at 20° C. The baNadD-1 02 1 02 1 bind to the aromatic site with their central cocrystals were obtained in conditions that contain 0.2-0.25 anthracene or benzene rings and hydrazone groups; while the 60 M magnesium formate and 20%-24% PEG 3350. The linker and the terminal chlorobenzene ring intrude into a deep baNadD-1 02 1 complex crystals were obtained from groove on the enzyme and interact directly with the conserved 0.2M potassium formate and 20% PEG 3350. Both crystals active site HXGH motif residues. In addition to this groove, were cryoprotected in Solutions that contained an increased the binding potential of a small pocket adjacent to the primary concentration of PEG 3350 (40%) and original components binding site is highlighted in the 1 02 1 and 1 02 3 com 65 of the reservoir and frozen in liquid propane. The baNadD plex structures, where it is revealed that this pocket favors 1 02 3 complex formed crystals in 0.2 M potassium citrate binding of a carboxylate group. In the 1 02, 3 complex and 20% PEG 3350, and the crystal was frozen in the cryo US 8,785,499 B2 39 40 protectant containing original components of the reservoir vo and v, represent initial rates in the absence and presence of supplemented with 10% DMSO and flash frozen in liquid inhibitors at concentration 1. nitrogen. Chemistry The X-ray diffraction data of the baNadD-1 02 complex crystal was collected at beamline 19BM, Advanced Photon 5 Proton NMR spectra were recorded on Varian 500MHz FT Source, Argonne National Laboratory, whereas the data for NMR spectrometers. Mass spectra were recorded on a LCQ baNadD-1 02 1 and baNadD-1 02 3 crystals were col mass spectrometer (Finnigan MAT, San Jose, Calif.). Ele lected in-house on a Rigaku FRE rotating anode X-ray gen ment analyses were performed by Atlantic Mircolab, Inc. erator equipped with Osmic focusing device and RAMS (Norcross, Ga.). Flash column chromatography was per IV++ image plate detector. The data were further processed 10 formed using Silica Gel 60 (230-400 mesh) from Thomas using HKL3000 software'. Scientific (Swedesboro, N.J.). Analytical thin layer chroma Both the baNadD-1 02 and baNadD-1 02 1 complexes tography (TLC) was performed on precoated glass backed were crystallized in the P222 space group, isomorphous to plates from Analtech Inc. (Newark, Del.) (TLC uniplates, the crystals of baNadD-3 02 complex reported recently''. Silica gel GHLF, 250LL). Plates were visualized using ultra Therefore, the model of the baNadD-3 02 (pdb code 3hfi), 15 violet, iodine vapors, phosphomolybdic acid or ninhydrin. excluding ligand and solvent molecules was used as the initial Compound 1 was available from commercial supplier. The model for the refinement of both new complexes using the purity of the compounds, as determined by GCMS, was program Refmac of the CCP4 package'''. The solution O5%. of baNadD-1 02 3 complex was found by the molecular replacement method of Phaser' using apo baNadD as the Synthesis of N-(2-Chloro-phenyl)-3-(4-3-(2- starting model. Model inspection and adjustment was per chloro-phenylcarbamoyl)-propionyl-hydrazonom formed with Coot". The electron densities for compound ethyl-benzylidene-hydrazinocarbonyl)-propiona 1 02, 1 02 1 and 1 02 03 were clearly visible in the early stage of the refinement. The PRODRG server' was mide (1 02 1, Scheme 1) (FIG. 15). used to generate the models for the compounds to be included 25 Benzene-1,4-dicarbaldehyde (0.01 g, 0.07 mmol) and in the complex structure. Final rounds of refinements were N-(2-Chloro-phenyl)-3-hydrazinocarbonyl-propionamide 1 performed using PHENIX'' ' and the model geometry (0.036 g., 0.14 mmol) in ethanol (5 mL) were heated to reflux was monitored by Molprobity". The crystal data and refine for 2h. After cooling to room temperature, the precipitate was ment statistics of these complexes are list in Table 14. The filtered off and washed with ethanol to give 1 02 1 as a pale coordinates have been deposited in the Protein Data Bank'' 30 white solid (0.03 g, 69%). H NMR (500 MHz, DMSO-d6) with accession codes 3MLA, 3MLB, and 3MMX. 12.31 (2H, s), 8.21 (2H, s), 7.94 (4H, s), 7.60-8.20 (8H, br): Enzyme Inhibition Assay MS Anal. Mol. Wt. 580.14 (604.2 M+Na). Elemental Analy A general phosphate detection assay method using Mala sis Calculated for C28H26C12N6O4 0.4H2O: C, 57.13: H, chite Green reagent was adapted to measure the activity of 4.58: N, 14.27. Found: C, 57.36; H, 4.49; N, 14.00. NaMN adenylyltransferase'". Briefly, the byproduct of 35 NadD catalyzed reaction, inorganic pyrophosphate (PPi), was hydrolysed by inorganic pyrophosphatase and the result Synthesis of (N'-tert-Butoxycarbonyl-hydrazino)- ing orthophosphate was detected by the Malachite Green dye. acetic acid ethyl ester (4, Scheme 2) (FIG. 16) The reaction mixture contained 2.3 nM ecNadD (or 1.2 nM baNadD) in 100 mM Hepes, pH 7.5 buffer, 0.2 mM ATP, 0.07 40 Ethyl bromoacetate 3 (6.97 mL, 62.8 mmol) was added to or 0.2 mM NaMN, 10 mM MgCl, 0.1 mg/ml Bovine Serum a stirred solution of tert-butylcarbazate 2 (24.9 g, 188.6 Albumin, 0.2 U inorganic pyrophosphatase. Appropriate mmol) in water (25 mL) at room temperature. The mixture amount of inhibitors were added to the reaction mixture to was stirred for 30 min. Water layer was then extracted with assess their effect on enzyme activity. After preincubation of ethyl acetate (3x). Ethyl acetate extracts were pooled together the enzyme with the compounds for 5 min at room tempera 45 and washed with brine. Ethyl acetate was evaporated under ture, the reaction was started by adding NaMN substrate. The vacuum to get crude product which was purified by flash reaction was quenched with two volumes of Malachite Green column chromatography using hexane:ethyl acetate (70:30) Reagent in 1.2 M sulfuric acid prepared as described by as an eluent (yield 70%). "H NMR (500 MHz, CDC1,) 1.28 Cogan et al.''. After 20-30 min incubation to allow for (3H, CH, CH, t), 1.45 (9H, C CH, s), 3.64 (2H, complex/color formation, the absorbance was measured at 50 NH-CH CO, s), 4.20 (2H, CHCH, q): MS Anal. Mol. 620 nm. To account for possible contribution of free phos phate and/or pyrophosphate (present in the sample or released Wt. 218.25 (M+1). due to non-specific hydrolysis of ATP) as well as of back ground absorbance (color) of the tested compounds, parallel Synthesis of 4-(N-tert-Butoxycarbonyl-N-ethoxycar reactions were run for each experimental point without addi 55 bonylmethyl-hydrazino)-4-oxo-butyric acid (5. tion of NadD enzymes, and their ODo values were sub Scheme 2) (FIG. 16) tracted from the measurements of enzyme activity in respec tive samples. Reaction in the presence of 2% DMSO but Into a solution of (N'-tert-Butoxycarbonyl-hydrazino)- without inhibitor served as the positive control. acetic acid ethyl ester 4 (1.85g. 18.5 mmol) in DMF (30 mL) For ICso determination, the initial rate of the enzymatic 60 was added Succinic anhydride (4.84 g. 22.2 mmol) and the reaction was measured at fixed NaMN and ATP concentra mixture was stirred at 75°C. for 18 h. DMF was evaporated tions (equal to two-fold K values) in the absence and pres and the crude mixture was purified by flash column chroma ence of various concentrations of inhibitors. The ICso value tography using hexane:ethylacetate (1% acetic acid) as an was determined by plotting the rates versus inhibitor concen eluent (yield 50%). "H NMR (500 MHz, CDC1,) 1.28 (3H, tration and fitting to the equation (I) using GRAPHPAD 65 CH, CH, t), 1.48 (9H, C CH, s), 2.55-3.00 (6H, PRISMOR). NH-CH CO, N CH-CH CO, m), 4.20 (2H, vivo (1+IIC.so) (1) CHCH, q): MS Anal. Mol. Wt. 318.25 (M-1). US 8,785,499 B2 41 42 Synthesis of{N-tert-Butoxycarbonyl-N-3-(2- Synthesis of{N-3-(2-Chloro-phenylcarbamoyl)- chloro-phenylcarbamoyl)-propionyl-hydrazino propionyl-N'-naphthalen-1-ylmethylene-hy acetic acid ethyl ester (6, Scheme 2) (FIG. 16) drazino-acetic acid (1 02 3, Scheme 3) (FIG. 17) Into a mixture of compound 5, HBTU and DIPEA in DMF naphthalene-1-carbaldehyde (0.015 g, 0.10 mmol) and was added 2-chloroaniline and the solution was stirred for 48 compound 8 (0.031 g, 0.10 mmol) in ethanol (5 mL) were h. DMF was evaporated under vacuum and the mixture was heated to reflux for 12 h. After cooling to room temperature, dissolved in ethyl acetate and washed with water (2x), 1M the precipitate was filtered off and washed with ethanol to KHSO (2x) and water (2x). Ethyl acetate was then evapo give 1 02 3 as a pale white solid (0.02g, yield 47%). H rated to get crude compound which was purified by flash 10 NMR (500 MHz, DMSO-d6) 2.64 (1H, N CH, CH, column chromatography using hexane:ethyl acetate (50:50) CO, t), 2.79 (1H, N CH, CH, CO, t), 3.17 (1H, N CH-CH CO, t), 3.23 (1H, N CH-CH CO, t), as an eluent (yield 38%). NMR (500 MHz, CDC1,) 1.27 (3H, 4.89-4.97 (2H, NH CH CO, m), 7.17 (1H, ArH, t), 7.31 CH, CH, t), 1.48 (9H, C CH, s), 2.58-3.06 (6H, (1H, ArH, t), 7.48 (1H, ArH, d) 7.56-7.68 (3H, ArH, m), 7.75 NH-CH CO, N CH-CH CO, m), 4.20 (2H, 15 (1H, ArH, d), 7.96-8.04 (3H, ArH, m), 8.50 (1H, ArNH, s), CHCH, q), 7.01 (1H, ArH, t), 7.22-7.27 (1H, ArH, m) 7.34 8.70 (1H, ArH, t), 9.56 (2H, Ar CH=N); MS Anal. Mol. (1H, ArH, d), 8.06 (1H, ArNH, s), 8.33 (1H, ArH, d); MS Wt. 437.11 (M-1). Anal. Mol. Wt. 427.88 (M+23). Other compounds may be synthesized in a similar manner as Scheme 2 (FIG. 16) by replacing the chemical precursors, Synthesis of{N-3-(2-Chloro-phenylcarbamoyl)- for example, replacing the 2-chloroanaline of Scheme 2 with propionyl-hydrazino-acetic acid ethyl ester (7. an unsubstituted aniline group or an aniline group Substituted Scheme 2) (FIG. 16) with at least one group X selected from halogen, hydroxy and alkyl. Compound 6 (0.3 g, 0.7 mmol) was dissolved in 5 mL of 20% TFA in dichloromethane and the solution was stirred for 25 REFERENCES 1 h. TFA was then evaporated under vacuum and the crude mixture was purified by flash column chromatography using All patents and publications mentioned in this specification ethyl acetate as an eluent (yield 87%). "H NMR (500 MHz, are indicative of the level of those skilled in the art to which CDC1,) 1.27 (3H, CH, CH, t), 2.75 (2H, N CH the invention pertains. All patents and publications herein are CH-CO, t), 3.11 (2H, N CH-CH CO, t), 4.20 (2H, 30 incorporated by reference to the same extent as if each indi vidual publication was specifically and individually indicated CHCH, q), 4.36 (2H, NH-CH CO, s), 7.00 (1'-1, ArH, as having been incorporated by reference in its entirety. t), 7.23 (1H, ArH, t) 7.33 (1H, ArH, d) 8.24-8.40 (2H, ArNH, 1. McDevitt, D., and Rosenberg, M. (2001). Exploiting ArH, m); MS Anal. Mol. Wt. 327.76 (M+1). genomics to discover new antibiotics. Trends Microbiol 9, Synthesis of{N-3-(2-Chloro-phenylcarbamoyl)- 35 611-617. propionyl-hydrazino-acetic acid (8) 2. Osterman, A. L., and Begley, T. P. (2007). A subsystems based approach to the identification of drug targets in bac Compound 7 was dissolved in 10 mL ethanol followed by terial pathogens. Prog Drug Res 64, 131, 133-170. addition of 1.2 mL of 1N NaOH. The mixture was stirred for 3. Gerdes, S.Y., Scholle, M. D., D'Souza, M., Bernal, A., 40 Baev, M. 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H., A Robotics and structure solution—from diffraction images to an ini Based Automated Assay for Inorganic and Organic Phos tial model in minutes. Acta Crystallogr D Biol Crystallogr phates. Analytical Biochemistry 1999, 271, 29-35. 2006, 62,859-866. Tables TABLE 1

Inhibitor: O

HN H C N O N1 N Sa O H F O 3 O2 102

Substrate: NaMN ATP NaMN ATP

K K K K (IM) C. (IM) C. (IM) C. (IM) C.

baNadD 184 2.4 325 5.5 2.3 102 2.9 ecNadD 259 2.5 219 2.4 7.2 5 - 1 7.1

US 8,785,499 B2 51 52 TABLE 2-continued ICs. (LM MICs. (LIM) Entry Compound structure ecNadD baNadD. E. coii B. anth B. subt 1511 O 191 98 <100° NA <50° R a NN H N O

TABLE 3 TABLE 5-continued protein Complex state PDB ID residues-in-site 2O Interaction (E) 15 27 -11.3 -35.8 ecNadD apo 1k4k GLY10, ASP109, SER18O 3 O2 -39.0 NaAD bound 1k4m HIS19, ARG46, ARG134 3 05 hsNMNAT-1 apo 1kku TYR55, ASP158, TRP169 35 NAD bound 1kqn LYS57, ASP158, SER222 25 321 NaAD bound 1kgo LYS57, ASP158, SER222 1 TAD bound 1kr2 LYS57, ASP158, SER222 baNadD apo 2dtm ASN39, MET109, SER 156 NaMN bound 2dtn ASN39, MET109, SER156 30 NaAD bound 2dtr LYS45, ASP108, SER156 TABLE 6 apo 3dv2 ILE21, ASN39, MET109 Attractive volW interaction energy NaAD bound 3e27 LYS45, ASP108, SER156 ID E. coi B. anthracis Human

35 1 O2 -80.4 -71.6 -58.2 1 03 -60.5 -84.1 -57.4 1 05 - 68.9 -104.7 -638 TABLE 4 1 11 -88.4 -71.9 -61.7 113 -58.0 -85.5 -52.4 ID E. Coi B. anthracis Human 115 -65.1 -83.0 -49.4 1 O2 -50.4 -44.2 -45.4 40 15 11 -63.1 -63.3 -47.8 1 03 -45.6 -46.6 -44.1 15 27 -60.4 -61.0 -46.3 1 O5 -47.7 -50.4 -44.3 3 O2 -64.3 -71.7 -54.8 1 11 -48.0 -42.1 -39.5 3 05 -39.5 -51.7 -48.8 113 -42.2 -58.3 -40.6 3 15 -70.8 -80.8 -42.7 115 -46.3 -51.8 -39.3 3 17 -71.8 -83.5 -51.O 15 11 -46.6 -45.6 -32.8 45 3 21 -61.7 -75.5 -48.1 15 27 -39.9 -40.4 -33.5 3 23 -76.2 -81.6 -43.3 3 O2 -37.2 -45.8 -31.9 3 C11 -80.9 -89.8 -49.9 3 05 -32.2 -30.8 -36.4 3 15 -40.2 -41.2 -31.7 3 17 -33.8 -31.9 -33.7 3 21 -43.2 -41.7 -3S.S 50 TABLE 7 3 23 -42.8 -48.1 -33.O 3 C11 -39.5 -44.2 -37.1 Data sets baNadDiproduct baNadD3 02 Data Statistics

55 SpaceSp groupgrou P2221-1- P2221 TABLE 5 Unit cell (A) a = 41.8, a = 88.79, Interaction (E b = 137.41, b = 97.53, interaction (E) c = 143.97 c = 44.30 Electrostati Wander Waals Resolution (A) SO-2.2 SO-2.0 (COS8C Total observations 157926 113171 ID E. coli B. anthracis Human E. coli B. anthracis Human 60 Unique Reflections 433S3 26467 1 O2 -11.6 -16.8 Completeness (outer shell) (%) 98.8 (89.9) 99.9 (100.0) O R.syn (outer shell) 0.085 (0.555) 0.037 (0.279)

o - Z. fö (outer shell) 15.9 (2.0) 36.8 (5.4) OX s Refinement 111 | 1.4 113 -13.0 Rivor” O.2O6 O.2OS 115 -11.9 65 R. O.276 O.266 15 11 -10.0 11.4 rim.S.d bond length (A) O.O11 O.O12 US 8,785,499 B2 53 54 TABLE 7-continued TABLE 7-continued

Data sets baNadDiproduct baNadD3 02 Data sets baNadDiproduct baNadD3 02 r.m.S.d bond angle () 1.49 1.44 ligands 24.4 41.1 Protein atoms 61.96 31 O2 5 water 39.9 34.4 Water molecules 494 309 Ramachandran Plot Ligand atoms 18O 53 Average B-factors (A2) Favored region (%) 97.0 98.7 Allowed region (%) 99.3 1OOO Protein 32.4 28.0

TABLE 8

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

OO1 SPECSNET AG-205 79% 88% 32429038 (X10) O

N1 N S H O I

OO2 MAYBRIDGE, RDROO32S C 76% 85%

S

2NNN ls N H H C C

003 SPECSNET: AM-807 7396 40% 13614404 (C11)

C C

OO4 CHEMDIV 8004-8936 O 71.9% 44% O.V /O. O O S2 e N O S.

O O

OOS MAYBRIDGE SEWO2O74 O 71.9% 44%

O06 SPECSNET AN-829, 62% 34% 13539519 Br H 21 N / N M US 8,785,499 B2

TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis OO7 CHEMBRIDGE 6766541 y H 54% 34% HN \ O

OH

O08 SPECSNET AG-690, C 47% O% 11635622 o={ C

N-()--O

O09 SPECSNET AK-968, O 45% O% 15608936 HO S y-/{ \ / Yp O O N /

O10 CHEMDIV S186-0398 H 32% 89% N N HO N-s 4. HO / n NH2

O11 CHEMBRIDGE SS371 OS C 31% O%

| Nty N S r O N&

O12 SPECSNET AG-690, N 30% O% 36106009

O13 SPECSNET AE-484 30% 32881023 n

C

O14 SPECSNET AN-023 30% 12769.011 US 8,785,499 B2 57 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coi B. anthracis

O15 CEHEMDIV C28S-0040 29% 57% (12B) F

HN HN Br O

O16 NANOSYN NS2925 OOC 29% 75% O

C NH NO

O17 CHEMBRIDGE, SS281.86 OH O 27%

OH O NN H O

O18 SPECSNET AK-918 27% 14550.057

O19 MAYBRIDGE CDOS223 26% 27%

O2O SPECSNET AK-918 26% 11592005

O O ) (O N-()-d S

O21 CEHEMBRIDGE SAO7857 24% 79%

C -----S S O O O M /S YNH,

O22 CEHEMBRIDGE S6S2726 24% 31% US 8,785,499 B2 59 60 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

O23 SPECSNET AG-2OS, \ 24% 34% 13109249 N M O N O Sn M O O N O n

O24 CEHEMBRIDGE 52386.75 O 23% O% OH \ ~~~ O O

F

O2S MAYBRIDGE 312870 23% 55%

O26 CHEMBRIDGE 6874722 23% O% HN Ns Nulls N NH

O

O27 SPECSNET AG-690 O 23% O% 36281062 NH \-N O OH O

O28 CHEMBRIDGE SA98.694 O 23% 79%

s N1 I H Nul) O

O29 SPECSNET AN-919, OH 22% O% 15527107 O O C

N O C US 8,785,499 B2 61 62 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

O3O CHEMBRIDGE S654,059 22% 28% (C3) c-( ) { OH

O31 CEHEMDIV C612-0726 21% 4%

O32 SPECSNET AN-465, Br 21% 8% 42768143

O33 CHEMBRIDGE S232616 HO OH

O34 CEHEMDIV 2023-OOS6 21% 196

O H N

O3S CHEMBRIDGE 791.6939

OH N OP

O36 SPECSNET AN-652 20% O% 41376,266 US 8,785,499 B2 63 64 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

O37 CHEMBRIDGE S376889 Br 20% O%

O HN O 2. S. \ S S OH

O38 CEHEMBRIDGE S357363 OH N 20% O% C N -S NH N N1

C

O39 SPECSNET AF-399, 20% O% 1SO3O248 Br NN -( N HN S. HO

Br O

O40 CHEMBRIDGE S38O358 Br 19% 20%

O HN N Y Y-N OH

O41 SPECSNET AG-690 O 19% 39% 33347062 O)-(N OH

C C O42 CEHEMDIV 8013-2042 j 19% n.d. d N > le C

O43 CHEMBRIDGE 5231111 18% 296

C US 8,785,499 B2 65 66 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

O44 CHEMBRIDGE S349303 OH 1796 O% Br Null N N le

C

O4S CHEMBRIDGE 6862662 1796 O%

O

HO N-OH I

O46 CEHEMBRIDGE 6431895 O 1796 71.9% H o-skyH N N-N Br

O47 SPECSNET AM-807 H 17% O% 43276008 N N O

S 21

C OH

O48 CEHEMBRIDGE 6604853 C O O 16% 14% \20 N \ H NH2

O49 CEHEMBRIDGE 66048O2 16% 10% H S S )/ry\ ro QO O N

OSO SPECSNET AF-399, O 16% 38% 15337214 \e O V NH2 Br N N H

O O

OS1 CHEMBRIDGE S604747 O 16% O% N1 NN R H O 2 NH2 HN N4 / US 8,785,499 B2 67 68 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

052 SPECSNET AI-204, OH 16% 24% 42879139 N O

O S HN C

O / C

053 SPECSNET AJ-292 OH 16% O% 422848.39 O 21 NN N N HO N S O

O54 CHEMBRIDGE 7921220 OH 15% 27% O N / NH2 OSS CHEMBRIDGE 64.1973O O-l 15% 15%

O56 SPECSNET AF-826 N.A. 15% 57% 30391 019

O57 CEHEMBRIDGE 6127O69 15% 28%

C

OS8 CHEMBRIDGE 77S1796 15% O%

S N CCO7 S O59 CEHEMBRIDGE 7936757 HN/ \ \ \, 15% O%

US 8,785,499 B2 71 72 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

O67 CEHEMBRIDGE S185703 O OH O OH 13% 196

21 O 21

O N N OH N N

OH O O68 CEHEMBRIDGE 60498.63 UC 13% 10%

N H

O69 CEHEMBRIDGE 6347634 O 13% 79% I N N HO S

^o O O

O70 CEHEMBRIDGE S680859 13% 59 O O

N O in- O \ O OH

O71 CEHEMBRIDGE 5917405 12% 79% H NS O.N/ N. NH S N \ Z

O72 SPECSNET AH-O34f H 12% O% 32474O10 N O O

N h \-0 H

O73 CHEMBRIDGE 7425408 o 12% O% O

O74 CEHEMBRIDGE S16O262 O 12% O% US 8,785,499 B2 73 74 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

O75 CEHEMBRIDGE 5914228 12% 3% O N. N

O76 CHEMBRIDGE S227209 12% O%

S-NH

O77 SPECSNET AG-205 12% O% 36696O20

O78 CHEMBRIDGE S173154 12% O%

O79 CEHEMBRIDGE 5237216 12% O%

O80 CHEMBRIDGE SS391.86 11% 24%

O81 CEHEMBRIDGE, SS49749 11% O%

O82 MAYBRIDGE DFPOOO19 11% S8%

O83 CHEMBRIDGE 7876O10 11% O% US 8,785,499 B2

TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

O84 CHEMBRIDGE 7798.274 11% C S V

N N H H F

O85 CEHEMBRIDGE 7528S98 11% 5-( O

O

O86 CHEMBRIDGE 7731718 11% HN OH C O C

O87 CEHEMBRIDGE S213908 Br 11% ~sOH H

O OH

O88 CHEMBRIDGE 791.4818 C 11% NN O 2 --- S S

N NN

O89 SPECSNET AE-848 HO O 10% 37% 3.3208046 O O

s NH HN OH N -) O

O90 CHEMBRIDGE 7785233 10% O HN

Yille N

- O O91 CHEMBRIDGE 7805704 N-(O Cl 10% N HN C US 8,785,499 B2 77 78 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis O92 CEHEMBRIDGE 5269569 / \ 10% O% / -N RN O

O93 CHEMBRIDGE S334483 O 10% O% \ HN ,

O N o1 NoSa O O94 CHEMBRIDGE 7593154 NX-s 10% O% O \ , 4' o22

095 CHEMBRIDGE 7502373 H2NNa O 10% O% S O % S W -NH2 HN W N O H

O96 SPECSNET AK-968, N.A. 10% 25% 15359755

O97 CEHEMBRIDGE 6160561 10% 63%

H r N O O

NH2

O98 CHEMBRIDGE 6381497 O OH 10% 13%

O O

O N NH

O H 1s.

O99 CHEMBRIDGE 659.2110 O 10% 296 \ 20

C US 8,785,499 B2

TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis 100 CHEMBRIDGE 7661577 c - O 9% 101 CHEMBRIDGE 7447806 C O 9% - -, i. t

102 CHEMBRIDGE SA28173 OH 9% 6%

103 CHEMBRIDGE 7907251 \ / 9%

104 CHEMBRIDGE S92S415 C 9% 29%

0. 105 CHEMBRIDGE 6652134 O S 21 9% 51%

N l N N N H H

F

106 SPECSNET AR-360 O 9% 42760781 / 1N N-N

107 CHEMBRIDGE, SS48185 O 9% OH O C

C N-K)—o US 8,785,499 B2 81 82 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

108 CHEMBRIDGE 768288O e 9% O% MY. Br

rt N H C

109 CHEMBRIDGE SA98423 9% 8%

110 CHEMBRIDGE 7883375 9% O%

OH

111 CHEMBRIDGE S67927O Br 9% 6%

N N N OH

112 SPECSNET AC-907. 8% O% 34131053 O \ Ns/ 113 CHEMBRIDGE 6111462 Cy 8% 12%

114 CHEMBRIDGE SSS3328 8% O%

OH 1n-1\ US 8,785,499 B2 83 84 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

115 SPECSNET AG-690, HO 8% O% 15432S42 O O N.N. O OSN' O

116 SPECSNET AN-46S O N 8% 59 14952.181 HO M N NH N M 7 n1 NN NY Y-N

117 SPECSNET AK-968, OH 8% 14% 371661.99 O O N I N1Y N N O

C

118 CHEMBRIDGE S323892 C 8% O%

C -K)--si|

119 CEHEMBRIDGES115114 O 8% O% OH O \ N N OH O

120 CHEMBRIDGES1171.15 O 8% O%

N1 N H O O OH

121 CHEMBRIDGE 7791534 OV-NH2 8% O% US 8,785,499 B2 85 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coi B. anthracis

122 CHEMBRIDGE 6081374 O 79% 6% OH O V O -( )- \-K O O HO

123 CHEMBRIDGE 7299424 O O 79% Ny A \ Br S

124 CHEMBRIDGE 7024854 79%

H N N NH / N SV N2 O

125 CHEMBRIDGE 727.04.09 79% NH HN ro O O O

126 CHEMBRIDGE 6480574 79% 22% N \ ( a N-\, o-/

127 CHEMDIV 4553-3701 OHH 79% 59% O H

O N o O - A. N \ N/ V H

128 CHEMBRIDGE 7845106 79%

129 SPECSNET AH-487. 79% 421.93471 US 8,785,499 B2 87 88 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

130 MAYBRIDGE NRBOO686 79% 28% O OH /

O HO O

131 CEHEMBRIDGE S227097 79% O% C N

Br O

132 CEHEMBRIDGE S320808 6% 25%

N

HO O

133 MAYBRIDGE SPOOOO1 N.A. 6% 38%

134 CHEMBRIDGE S186398 6% O% OH O OH

O N

O HO

135 CEHEMBRIDGE S2O1899 H 6% 10% N OH N

C

136 SPECSNET AG-690 O HO 6% 64% 4O246195 S HN O M NH HN

137 CHEMBRIDGE 67347OO 6% 79% US 8,785,499 B2

TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis 138 CEHEMBRIDGE S934320 p 6% S4% o2N W S N N2. NH2 O

139 CEHEMBRIDGE 7876477 HN / 6% O% NH O O O2 sV O Cl

140 CHEMBRIDGE 7929927 N 6% O% yN-( O S 4. O / SNH,n

141 MAYBRIDGE CC184O1 N 6% 1796 e N N COO S.

142 CEHEMBRIDGE S175596 6% O% H O N- S\ S{ull-NM O

143 CHEMBRIDGE 5621SSS S N 6% 296 Q N- \, KN H N

144 CHEMBRIDGE 6449068 O 6% 65% OH O

145 SPECSNET AN-988. 6% 42879.152

146 CHEMBRIDGE S9071.93 59% 11%

C US 8,785,499 B2 91 92 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

147 CHEMDIV 2181-0361 OH 59% 12%

O N uC H ChrSa n I N

148 MAYBRIDGE KMO8231 59% 4%

O 149 CHEMBRIDGE 7744903 O\1\ 59% O% \, /NN ly1 \ N2 NH2

150 SPECSNET AN-465, 59% O% 428.87914 \, { ) O

? -K)--O

151 SPECSNET AQ-911, O 59% O% 424.64333 21 N

OH O

152 CHEMBRIDGE S221975 O 59% O% OH O C

N

153 CHEMBRIDGE 6999589 HO 59% O% \ N O \,

N N \ . O OH

154 CHEMBRIDGE 76935S2 59% O% US 8,785,499 B2 93 94 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis 155 CHEMDIV 3820-2901 O)-(O 59% 26% S N-(N-( N S

156 SPECSNET AA-516, 59% 22% 25O12123 7

S

O HO O

157 SPECSNET AN-46S C C 59% O% 4288.8516

N S-NH ? O

158 MAYBRIDGE KMO2656 4% 36% O N. I

8 || | OH Ns S

O

159 NANOSYN NS33821 QH N2\S 4%0. 1960. N S.

COO

NO

16O SPECSNET AG-690, N-N 4% O% 13508068 OH N S H O

161 MAYBRIDGE SEWOO3S1 NH 4% 68% - n S. N C

162 CEHEMDIV 0783-O142 HN 4% O% O F ( ) -N) K ) US 8,785,499 B2 96 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

163 CHEMBRIDGE 6229693 C 4% S4% Br H 1 N

O

164 CHEMDIV 458S-OO16 4% 32%

O

16S CHEMBRIDGE S467380 O 4% O%

C N-- O O \ 1N1)-1 H H

166 CHEMBRIDGE S227796 OH 4% 19%

NN O

167 SPECSNET AF-399, 4% O% 15335566

O

168 SPECSNET AG-690, OH 3% O% 15444484

C

169 CHEMBRIDGE 7842136 3% O% US 8,785,499 B2 97 98 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

170 CHEMBRIDGE 7697135 3% O% HN

O s'Sa / OIso \N

171 SPECSNET AK-968, O OH 3% 24% 41026368 O N1 NO H O O

172 SPECSNET AN-989, HO O 3% 32% 14834.030 O

O 7N- N \

Br 173 CHEMBRIDGE 7741517 i-JO 3% O% O 2N SKNH NH2

174 SPECSNET AG-2OS, O&O 3% 11% 32366049 Nt O ICl. N F H O

OH

175 CHEMBRIDGE 7579538 C HO 3% O% HO O O

176 SPECSNET AN-46S O 3% O% 1540 1042

N ls OH NH - O

177 CHEMDIV 8006-2677 O 3% 21% US 8,785,499 B2 99 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

178 CHEMBRIDGE 784.2469 O 3% O% 'Ni O O O O v 4 V Nt C O O/

179 SPECSNET AO-080. O 3% O% 42479871 --

a vO S-NH

O

180 CHEMDIV S983-3833 3% 15%

N S O

r2N OH

181 CHEMBRIDGE 6047SSO OH 3% 56%

O O O N1

HO O

O OH HO O

182 CHEMDIV 1889-33.25 3% 14% s V

O OH

183 CHEMDIV 7213-0565 C 3% 79%

N N- N s HN O \,N

184 CHEMBRIDGE 67231.83 O O 2% 16%

OH US 8,785,499 B2 101 102 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis

185 NANOSYN NS8477 O 2% 40%

C O

186 CHEMDIV 1831-O153 C 2% 9% O N NS O M S HN \ / vW O

187 CHEMBRIDGE 7633313 \ 2% O% HN NN

N OH Br

188 CHEMBRIDGE 731 6103 S 190 O%

O) \in- Br O

189 CHEMDIV 2389-1926 O O 190 O% sus Br

O1. N1 N N N H

190 SPECSNET AQ-360/ 1. 190 O% 416.15677 NuN O O OH

191 MAYBRIDGE SEWOS479 C 190 59

2N.

C US 8,785,499 B2 103 104 TABLE 8-continued

INHIBITION

# VENDOR ID STRUCTURE E. coii B. anthracis 192 SPECSNET AG-690, C O- 190 O% 15439214

Oi-S O OH 193 CHEMBRIDGE 5220945 / \ 190 18% N

o S

O HO

194 CHEMDIW 4361-0771 HN O OH 190 62%

O={ N1S H I

195 CHEMBRIDGE 5571553 O 190 O%

HN

NH C

O HO O

196 CHEMDIV C563-0504 - O 190 52%

v O in- N O YNullO OH

197 CHEMBRIDGE 6341,126 N O% 34% )—s O N \ 4 N H HN- / NNJ S

198 CHEMDIV 3966-3S28 O NH O% 79% X=NH S

C

199 SPECSNET AQ-390/ O O% O% 14195074

OH C S-NH US 8,785,499 B2 105 106 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coi B. anthracis

200 CHEMBRIDGE 6048997 Br 76%

HN

H O N-N

201 CHEMDIV 5618-4578 25%

2O2 MAYBRIDGE BTBOO374 32%

2O3 CHEMBRIDGE 780O297

204 CHEMDIV O28S-OO72

2OS CHEMDIV SO24-0069 40%

206 CHEMDIV S743-0118 25%

N

2O7 CHEMDIV 221 6-OOO1 79% OH US 8,785,499 B2 108 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

208 CHEMBRIDGE 72O7373 F

7& S N 4. \,

209 CHEMBRIDGE 77291.04 OH

C O

N H C HO

O

210 CHEMDIV 8O12-2515 N.A.

211 CHEMDIV 2112-OOO7 OH

O| / NH S-N

O

212 CHEMBRIDGE 66.58496 O O

S-NH OH

O

213 CHEMBRIDGE 6181516 O O H.N.N. N O H

214 CHEMDIV 1636-O418 O

N SEO / Br NH2

21S CHEMDIV 3453-1439 N.A.

216 CHEMDIV S186-0454 H Y N COO RN OH

OH HO US 8,785,499 B2 109 110 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

217 CHEMDIV C301-2383 C

N r C

HO O

O

218 CHEMDIV 7009-0719 O \-NH2S O M

H CCO O 219 CEHEMDIV 3583-1608 HN N NN 2- \ / V o N N-1- s

HN

220 CHEMDIV 7009-072O O

221 CEHEMBRIDGE 6464120

222 SPECSNET AN-329 43211,385

223 CHEMBRIDGE S2324.80 US 8,785,499 B2 111 112 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

224 CEHEMDIV 8O15-2343 N 21 S N 1. H H S N

225 CHEMBRIDGE 6154949 Br

HN N

2 N-N N

226 CHEMDIV 8009-1988 N.A.

227 CHEMDIV 1611-4019 O

S-N | O o=y O \,, HO

228 CHEMDIV S330-0093

229 CEHEMDIV 8008-701S S -

230 CHEMDIV 1218-2052

O

231 CEHEMDIV 6456-0640 O / t S HN Sl

232 CEHEMDIV 3448-4094 US 8,785,499 B2 113 114 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

233 CHEMDIV 8013-2S25 8% NH / O% A. , C. NEO N O M O

234 CEHEMDIV 3616-0014

O \ / OH

23S CHEMDIV 7296-3325 OH

N O O N N N / OH H N H

236 CHEMBRIDGE 7518597 OH O O

NH S -()

HO O

237 CHEMBRIDGE 7942923 HN

s N C N Ns

238 CEHEMDIV 6049-1958 O 14%

N "-- H

OH

239 CEHEMDIV 3552-0822 O 11%

H O

O to f S \ N-N

240 CHEMDIV O917-O113 US 8,785,499 B2 116 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

241 CEHEMBRIDGE 5927580

242 CEHEMDIV 3098-0089

243 CHEMDIV CS15-2290

244 SPECSNET AN-329 C 41508710 C

H H

24S CHEMBRIDGE 7827373 HO N-(O Cl HN Br

246 CEHEMBRIDGE S321,518 O

S-NH

O OH

247 CHEMDIV 4227-2396

248 CEHEMDIV 2806-0043

US 8,785,499 B2 121 122 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

26S CHEMDIV C324-1252

266 CEHEMDIV O801-1033

267 CEHEMDIV 4896-34O2

N N OH H

268 CEHEMBRIDGE S4690SO O Ye\ 2O NH2 O O V NR N N NH2 H

269 CEHEMBRIDGE 66765.15 NH2 OESEO O

270 CEHEMDIV 3973-0479

271 CEHEMDIV C614-1037

272 CEHEMBRIDGE 6615918 HN N - le N

273 CHEMDIV O242-0575 N.A.

US 8,785,499 B2 125 126 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

28S CHEMDIV C224-4321

286 SPECSNET AG-690 4O753590

287 SPECSNET AM-807 13614710

288 CHEMBRIDGE, SS43949

289 CEHEMDIV C224-2785

290 CHEMBRIDGE 7905975

OH

291 CHEMDIV 1813-1087

292 CEHEMDIV 4694-0709

US 8,785,499 B2 129 130 TABLE 8-continued

INHIBITION

i VENDOR ID STRUCTURE E. coii B. anthracis

301 CHEMDIV

O / N" Br o-N

302 SPECSNET AG-690 N 11667663 O / \

N HN

O O

303 SPECSNET AE-848 O 3.3221011 O OH

O O

O OH

304 SPECSNET AN-329 HO 41189553 O

N HN 2 SN N. O O1. N O H

305 SPECSNET AK-968, 121 OOO46 C N HN -{N-NH

306 SPECSNET AN-652 O H 413093O2 Y- N S N

C Or

307 SPECSNET AO-081, 417563S4

Br US 8,785,499 B2 131 TABLE 9

INHIBITION (%) ICso (LIM)

VENDOR ID STRUCTURE E. coii B. anthracis

SPECS AM-807 7396 40% 13614404 (C11)

CHEMDIV 5350-0377 100% 100% 25 25

CHEMDIV 5350-0029 98% 100% 65 36

SPECS AM-807 93% 90% 13614405 64 21

O3 04 SPECS AM-807 84% 79% 3614701 21 21

O3 05 SPECS AM-807 81% 90% 13614315 2O 9

SPECS AM-807 80% 40% 13614765 >2OO >2OO

US 8,785,499 B2 135 TABLE 9-continued

INHIBITION (%) ICso (LIM)

VENDOR ID STRUCTURE E. coii B. anthracis

O3 14 CHEMDIV 46% 92% 117 38

O3 15 SPECS AM-807 46% 87% 13614362 51 12

O3 16 SPECS AM-807 46% 83% 13615708 50 35

O3 17 SPECS AM-807 44% 64% 13614744

O3 18 SPECS AM-807 41% 71.9% 13614342 35 14

O3 19 SPECS AM-807 32% 61% 13615674

O3 20 SPECS AM-807 24% 11% 14147089 >2OO >2OO US 8,785,499 B2 137 138 TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis

O3 21 CHEMBRIDGE 7904181 22% S8%

O3 22 SPECS AM-807 21% 83% 13614667 1OO 39

O3 23 SPECS AM-807 20% 75% 13615675 >2OO 63

O3 24 SPECS AM-807 9% 59% 13615668 >2OO >2OO

O3 25 SPECS AM-807 8% 190 1274O16S

O3 26 CHEMDIV 5350-0047 79% 23%

03 27 SPECS AM-807 O% 20% 13614751 US 8,785,499 B2 139 140 TABLE 9-continued

INHIBITION (%) ICso (LIM)

i VENDOR ID STRUCTURE E. coii B. anthracis

O3 28 SPECS AM-807 O% O% 13614758 C

C

O3 29 SPECS AM-807 O% O% 13614745

C

OO1 SPECSNET AG-205 79% 88% 32429038 15 21 (X10) O N1 NN. H O I

O1 O1 CHEMDIV 8005-4955 90% 81% 6 4

O

B r N1 N S H O

O1 O2 CHEMDIV 8003-9695 80% 1.01% 15 25

O 1'sN H O

O1 O3 CHEMDIV 8003-6329 80% 97% 18 33 N 4 r-r NN O O Br US 8,785,499 B2 141 142 TABLE 9-continued

INHIBITION (%) ICso (LIM)

i VENDOR ID STRUCTURE E. coi B. anthracis

O1 O4 CHEMDIV 8003-88SO 79% 100% 15 25

Br O

N1 N S H O

O1 O5 CHEMBRIDGE 6048997 O 78% 88

Br

O1 O6 CHEMDIV 1761-1053 Br 78% 54%

O1 07 CHEMDIV 1761-1076 779, 81%

O1 O8 CHEMDIV 1761-0662 Br 75% 74%

O1 O9 CHEMDIV 1761-0639 Br 74% 68%

O1 10 CHEMDIV 8006-3094 729% 53%

Br

Br US 8,785,499 B2

TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis

O1 11 CHEMDIV 1761-0064 Br 70% 86% 30 1.

2 Br N NN H O 1.

O112 CHEMDIV 1761-0644 68% 75% O 48 N1 NN1 N. H O Br

O1 13 CHEMDIV 1761-0615 67% 29% O 170 N N1 N. Br H O Br

O1 14 CHEMDIV 1761-0686 O 60% 70%

N1 N S. H O

O1 15 CHEMDIV 1761-0634 F 47% 22% O 122 N1 NN. H

Br

O116 CHEMDIV 1761-0591 OH 42% 29% O N1 NN. H O F

O1 17 CHEMDIV 8005-4949 I 41% 35% O B r N1 NN H

O1 18 CHEMDIV 1761-0651 O 38% 49% H N Br

N Br O St US 8,785,499 B2 145 146 TABLE 9-continued

INHIBITION (%) ICso (IM)

VENDOR ID STRUCTURE E. coi B. anthracis

O15 CHEMDIV C28S-004O 29% 57% (12B) F 30 16 Br e N HN H O

15 O1 CHEMDIV C28S-0041

C

15 O2 CHEMDIV C28S-0043 20% 29%

Z N

N O

15 O3 CHEMDIV 1996 27% --OrNd

15 04 CHEMDIV C28S-0042 18% 26%

Z N

N O

15 05 CHEMDIV C28S-0047 1796 23% CrCa NN sN

15 06 CHEMDIV 15% 49% US 8,785,499 B2

TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis 15 O7 CHEMDIV 5682-O155 Ol 15% 45% a NN C H

HN O

15 O8 CHEMDIV C285-0037 14% O%

21 NN H O N no O \

15 O9 CHEMDIV C285-0027 Br 13% 60% O >2OO 111 a NN H

HN O

15 10 CHEMDIV 5682-0153 13% 26%

a NN H

HN O

15 11 CHEMDIV C285-O115 O 12% 63% 191 98 K a NN H N O

15 12 CHEMDIV 5682-0015 SN 12% 25%

Z NH

N O H

15 13 CHEMDIV C285-0028 C 12% 36% US 8,785,499 B2 149 150 TABLE 9-continued

INHIBITION (%) ICso (IM)

VENDOR ID STRUCTURE E. coi B. anthracis

15 14 CHEMDIV 12% 70% 88 74

15 15 CHEMDIV 12% 10%

1516 CHEMDIV S682-0013 11% 6%

15 17 CHEMDIV 11% 22%

15 18 CHEMDIV 11% 24%

15 19 CHEMDIV C28S-0033 11% 14%

15 20 CHEMDIV S682-0012 11% 8%

N O US 8,785,499 B2 151 152 TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis 15 21 CHEMDIV C285-0070 10% 30%

15 22 CHEMDIV 5682-O157 10% 28% F F e N HN H

15 23 CHEMDIV C285-0071 10% 32%

15 24 CHEMDIV C285-0023 9% 1796 21 NN H

HN O

15 25 CHEMDIV C285-0050 9% 16%

15 26 CHEMDIV 5682-0149 8% 8% Br

e N HN H

15 27 CHEMDIV C285-0035

15 28 CHEMDIV C285-O112 8% 24% >2OO >2OO

21 N H

N O US 8,785,499 B2

TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis 15 29 CHEMDIV C285-0107 O 79% 38% a NN H

HN O

15 30 CHEMDIV C285-0078 Crs 79% 42%

21 NN H

HN O

15 31 CHEMDIV 5682-0010 79% 27% 118 112

Z NH

N O H

15. 32 CHEMDIV 5682-0009 70, 4% H

OCN O H

15 33 CHEMDIV 5682-0159 JO 6% 15% 21 NN H

HN O

15 34 CHEMDIV 5682-0160 1s 4% 9% 21 NN H

HN O

15 35 CHEMDIV C285-0072 4% 19% O a NN H N O 15 36 CHEMDIV 5682-0154 Q 4% 11% US 8,785,499 B2 155 156 TABLE 9-continued INHIBITION (%) ICs (LIM

i VENDOR ID STRUCTURE E. coii B. anthracis 15 37 CHEMDIV 5682-0148 4% 190

e N HN H O O /

15 38 CHEMDIV 5682-0074 H 3% 1796 N

O N H

15. 39 CHEMDIV 5682-0077 3% 18%

21 N H

HN O

15 40 CHEMDIV C285-0108 196 O%

21 NN H OH N no O

15 41 CHEMDIV C285-OO76 O% 13%

a NN H

HN O

15 42 CHEMDIV 5682-0144 O% 11%

O HN

O

TABLE 10 NMNAT X10 chemdivO39751 similarO4 COMP NAME IDNUMBER MW logP 100 1 uCl O X10 chemdivO39751 8OOS-495S 474 6.17 1 Br N 1- N NN 2 S O US 8,785,499 B2 158 TABLE 10-continued

NMNAT X10 chemdivO39751 similarO4 COMP NAME IDNUMBER logP

2 chembridge0164358 6048997 438 4.84

Br

N

N-N O

O

3 O O M o chembridge018767 5247971 409 4.29

-( )- Br

C

4 chembridge048642 S478006 374 3.66

N -N N Br N-Q O O

5 I chembridge048.881 5479078 500 4.85

N -N N Br n-Q O O

6 chemdivO24765 8001-3249 450 S.28

Br -O N NN N O O

7 N N Br chemdivO284O6 8002-290S 467 4.94 n- N Br O O r

8 N NS 2 chemdivO28415 8002-2934 388 4.1 n- N O O Br US 8,785,499 B2 159 160 TABLE 10-continued

NMNAT X10 chemdivO39751 similarO4 COMP NAME IDNUMBER logP 1OO

chemdiv0300479 8001-92.57 413 S.49

10 chemdiv0303607 8003-88SO 474 6.14 10

Br

11 C chemdiv0303616 8003-8866 409 4.29

12 chemdiv0304370 8004-3740 467 4.97

Br

Br

13 chemdiv031240 8003-0754 409 4.29

Br

C

14 chemdiv032918 8003-6329 488 6.58 14

Br US 8,785,499 B2 162 TABLE 10-continued

NMNAT X10 chemdivO39751 similarO4 COMP NAME IDNUMBER MW logP

15 O chemdiv034066 8003-969S 430 5.97 15 C N 1- N NN 2 O S

16 chemdiv034267 8004-0194 453 4.49 N O Br O N-N

17 Na 2 chemdiv039712 8OOS-4902 374 3.7 N

O Br N O

18 Na 2 C chemdiv039718 8OOS-4908 409 4.33 N

O Br N O

19 N 2 chemdiv03.9747 8OOS-4949 500 4.89

O Br N O I

2O chemdiv043228 453 4.53 N O Br O

Br

21 chemdiv103114 1761-0568 392 3.85 21 Br

F

N 7