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Discovery of small molecule probe that shows anti-tubercular activity via Mtb bioA (DAPA synthase) enzyme inhibition
Dominick Casalena, Partha P. Nag, SaeWoong Park, Daniel Wilson, Rahul Edwankar, Stephen Johnston, Hanh Le, Roger Schilling, Joshua A. Bittker, Sivaraman Dandapani, Benito Munoz, Ran Dai, Barry C. Finzel, Dirk Schnappinger, Courtney Aldrich, Stuart L. Schreiber, and Michelle Palmer.
Author Information and Affiliations
Received: April 15, 2014; Last Update: January 16, 2015.
Cofactor biosynthetic pathways are a rich source of potential bacterial drug targets because of their essential nature and lack of corresponding mammalian pathways. The genome sequence of Mycobacterium tuberculosis (Mtb) H37Rv, the model virulent laboratory strain, revealed it has the full metabolic potential to synthesize all vitamins and cofactors. Biotin (vitamin H) is the cofactor responsible for activation of carbon dioxide in acyl-CoA carboxylases involved in fatty acid metabolism and pyruvate carboxylase in gluconeogenesis. Biotin biosynthesis is required for survival of Mtb in vitro and in vivo. Moreover, biotin starvation of an Mtb biotin auxotroph led to cell death, which is unusual as most Mtb auxotrophs enter a nonreplicating phase when starved for their given nutrient in vitro. In a recent study, Schnappinger and co-workers have demonstrated, using a phenotypically well-regulated bioA knockdown Mtb mutant, that silencing of bioA (DAPA synthase) after establishing an infection can defeat a chronic infection in a murine model of TB. These genetic studies provide an unprecedented level of validation for a Mtb target and suggest biotin biosynthesis, BioA in particular, represents an extremely attractive target for the development of new antitubercular agents. We report the discovery of a structurally novel small molecule (CID 1245700) that inhibits bioA and demonstrate antitubercular activity in wild type and Mtb strains that conditionally express bioA.
Assigned Assay Grant No: R03 MH096537-01
Screening Center Name & PI: Broad institute Probe Development Center, Stuart L. Schreiber, PhD
Chemistry Center Name & PI: Broad institute Probe Development Center, Stuart L. Schreiber, PhD
Assay Submitter & Institution: Courtney Aldrich, PhD / Dirk Schnappinger, PhD, University of Minnesota and Weill Cornell Medical College
PubChem Summary Bioassay Identifier (AID): 623896
Probe Structure & Characteristics
ML406
| CID/ML No. | Targets | BioA_IC50 (μM) [SID, AID] | WT M. tb (H37Rv) growth inhibition without biotin_IC50 (μM) [SID, AID] | Anti-Target | Fold Selective* |
|---|---|---|---|---|---|
| CID 1245700 ML406 | BioA | 0.03 [SID 164225891, AID 743357] | 3.2 [SID 164225891, AID 743070] | BioD | >100× |
- *
Selectivity = Anti-target IC50/Target IC50
1. Recommendations for the scientific use of the probes
Mycobacterium tuberculosis (Mtb), the primary etiological agent of tuberculosis (TB), has plagued mankind for millennia and remains the leading cause of bacterial infectious disease mortality.1-3 Biotin is an essential cofactor in Mtb and required for the establishment and maintenance of a chronic infection. The goal of the project is to discover a small molecule inhibitor of BioA, an enzyme involved in biotin biosynthesis in Mtb that may serve as lead compounds for drug development for TB. Additionally, rapid acting small-molecule inhibitors can be used as tool compounds to reveal mechanistic insights into the biotin-starvation induced death of Mtb, including potential synergistic effects with other therapeutic pathways.
2. Materials and Methods
Materials and Reagents
- Fluorescent dethiobiotin (Fl-DTB): Synthesized at C. Aldrich lab, University of Minnesota.
- E.coli BioD: bioD gene was PCR amplified from E. coli BL21 (DE3) genomic DNA and cloned into pET28b to introduce an N-terminal HIS tag. Overexpression of BioD was performed in E. coli BL21-CodonPlus (DE3) cells, and purification of the soluble protein was performed via Ni-NTA affinity chromatography to afford 420 mg of BioD per liter of culture.
- M.tuberculosis BioA: bioA gene was PCR amplified from M. tuberculosis H37Rv genomic DNA and cloned into a pET vector to introduce an N-terminal HIS tag then subcloned into pUC18 to create a vector under control of the lac promoter following the strategy described by Mann and Ploux.13Overexpression of BioA was performed in E. coli BL21-CodonPlus (DE3)-RP cells, and purification of the soluble protein was performed via Ni-NTA affinity chromatography to afford 1.5 mg of BioA per liter of culture.
- 7-keto-8-aminopelargonic acid (KAPA) HCl: Synthesized by the C. Aldrich lab, at the University of Minnesota in five steps from Boc-L-alanine using several modifications to the described synthetic route by Lucet and co-workers24 and obtained as the hydrochloride salt in 85% yield and an enantiomeric ratio of 98:2 as determined by chiral derivitization and HPLC analysis.
- 7,8-diaminopelargonic acid (DAPA) stock solution: synthesized by the C. Aldrich lab, University of Minnesota, as described (Vasantkumar, G.R.; Bhor, V.B.; Surolia, A. Syn. Commun. 2007; 37: 2633-2639.)
- Mtb H37Rv, Mtb bioA TetON-1, and Mtb bioA TetON-5 have been described in PMID 219802888
2.1. Assays
2.1.1. Primary assay – Coupled fluorescent dethiobiotin displacement assay for BioA inhibition
BioA catalyzes the reversible transamination between KAPA and DAPA. In the second step, which is irreversible, BioD catalyzes the ATP-dependent carbonylation of DAPA to provide dethiobiotin (DTB) and this step drives the BioA reaction forward (1A). Dethiobiotin is detected by displacement of the fluorescent dethiobiotin probe (Fl-DTB) from streptavidin resulting in an increase in the fluorescent signal. The essence of the assay lies in the fluorescence quenching of Fl-DTB by streptavidin and restoration of fluorescence upon release from streptavidin. Another critical feature of the assay that enables it to be performed in a continuous format is the rapid reversible binding of dethiobiotin and Fl-DTB as a result of the substantially weaker binding affinities of the molecules for streptavidin (Kd(App)=9.5 nM), which contrasts with the functionally irreversible binding of biotin. No increase of fluorescent signal indicates inhibition of dethiobiotin synthesis, and a positive result.
Compounds and controls were added at 10mM in DMSO (7.5nL) to 1536 well assay plates. Substrate (KAPA) solution, target enzyme (BioA and BioD) solutions, and fluorescent probe (Fl-DTB) quenched by streptavidin were added to the compound wells. After 45 minutes the BioA and BioD catalyzed synthesis of dethiobiotin reactions were quenched with EDTA and fluorescence reads were taken on a Viewlux plate reader (Ex 485, Em 530). Primary HTS data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO treated samples and the positive control (10 μM CHM-1, CID 357860). For the HTS, the average of two replicates was used to rank order activity and to choose compounds for retests. For dose studies, percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener's Condoseo software module.
2.1.2. Secondary assay – Coupled fluorescent dethiobiotin displacement assay for BioD inhibition
This counter screen identifies compounds that are inhibitors of the downstream enzyme BioD in the biotin synthetic pathway. The assay uses the same fluorescent probe (Fl-DTB) to detect inhibition of dethiobiotin synthesis as the primary screen. Here the BioA enzyme and KAPA substrate are eliminated from the assay. DAPA is introduced as the starting substrate and inhibition of enzyme activity, detected as no increase in fluorescent signal, indicates compound inhibition of the enzyme BioD. Compounds that are active in this assay are eliminated from further studies since they are targeting the downstream enzyme BioD instead of BioA or are possibly fluorescence quenchers.
Compounds and controls were added at 8-point dose in two fold dilutions from 10 - 0.78 mM in DMSO (7.5 nL) to 1536 well assay plates. Substrate (DAPA) solution, target enzyme (BioD) solution, and fluorescent probe (Fl-DTB) quenched by streptavidin were added to the compound wells. After 45 minutes the BioD catalyzed synthesis of dethiobiotin reactions were quenched with EDTA and fluorescence reads were taken on a Viewlux plate reader (Ex 485, Em 530). All values were normalized against DMSO treated samples and the positive control (EDTA added to positive control wells prior to substrate addition). For dose studies, percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener's Condoseo.
2.1.3. Secondary assay – PLP Cofactor Activity counter screen assay
In this counter screen assay compound inhibition of the enzyme cofactor Pyridoxal-5′-phosphate (PLP) is determined by monitoring a decrease in NADH absorbance at 340 nm. Aspartate Transaminase (AST), also known as serum glutamic oxaloacetic Transaminase (GOT) or aspartate aminotransferase (ASAT/AAT), facilitates the conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate. PLP is a co-enzyme to AST in this transamination reaction. Oxaloacetate and NADH are then converted to malate and NAD by the enzyme malate dehydrogenase. The decrease in NADH absorbance at 340 nm is proportionate to AST and PLP cofactor activity. Absorbance readings are taken at time 0 and 10 minutes. Compounds with inhibition activity to the enzyme and PLP cofactor will result in no NADH absorbance decrease in the assay. Compounds that are active in this assay are eliminated from further studies since they display activity against a functionally related PLP-dependent enzyme and are thus unlikely to possess useful selectivity.
2.1.4. Secondary assay – Mammalian cell cytotoxicity assay
Cytotoxicity assays are widely used by industry to screen for cytotoxicity in compound libraries. Cytotoxic compounds often yield a false positive result from the initial high-throughput screen and thus need to be filtered out to prevent investing significant time and expense during development of a potential probe.
Three cell lines representing in-vivo process were chosen for this in-vitro panel:
- HepG2, as human liver cells
- HEK293, as human kidney cells.
- A549, as a human lung cancer cells.
Compounds identified as active will be toxic to cells at a compound concentration less than 10 μM. Activity in the assay leads to a reduction in cellular ATP levels which correlates with a decreased luminescence signal from the read reagent (CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit no cytotoxicity at < 20 μM in all cell lines tested will be prioritized for additional studies.
2.1.5. Whole cell growth inhibition (Mycobacterium tuberculosis)
We measured to what extent small-molecule inhibitors of the enzyme BioA (7, 8-diaminopelargonic acid synthase) impaired growth of wild-type (WT) Mycobacterium tuberculosis H37Rv (Mtb), Mtb bioA TetON-1 (SD1 strain), and Mtb bioA TetON-5 (SD5 strain). Mtb bioA TetON-1/5 are mutants in which expression of BioA is induced by anhydrotetracycline (atc) (PMID 21980288). In the presence of atc Mtb bioA TetON-1 (SD1 strain) expresses ∼12× more BioA than WT and Mtb bioA TetON-5 (SD5 strain) expresses approximately 5× less BioA than WT. Growth of Mtb bioA TetON-1 and Mtb bioA TetON-5 was analyzed in media that contained atc but was free of biotin. Growth of WT Mtb was analyzed in biotin-free and biotin-containing media.
Small molecules that impair growth of Mtb specifically due to inhibition of BioA were expected to be only active in biotin-free media and in these media to be most active against Mtb bioA TetON-5 (SD5 strain) and least active against Mtb bioA TetON-1 (SD1 strain).
2.2. Probe Chemical Characterization
The probe (ML406) was synthesized as described in Section 2.3 and was subsequently analyzed by UPLC, 1H and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy are consistent with the structure of the probe, and UPLC indicates an isolated purity of greater than 95%. The physical properties of the probe ML406 are summarized in Table 1. The solubility of ML406 was determined to be 88.0 μM in PBS with 1% (v/v) DMSO (PBS; pH 7.4, 23 °C).
Table 1
Summary of Probe Properties Computed from Structure ML406.
The chemical stability of the probe ML406 was confirmed in the presence of PBS pH 7.4 with 1% DMSO. The probe ML406 was added (in triplicate at 1 μM) on six separate plates and allowed to equilibrate at room temperature for 48 hours. At each time point (0, 2, 4, 8, 24, and 48 hours), one plate was removed and an aliquot was taken out from each well and analyzed by UPLC-MS. After 48 h, more than 80% of ML406 remained (Figure 1).
Figure 1
Stability of the Probe (ML406, CID 1245700) in PBS Buffer (pH 7.4, 23°C).
The probe ML406 is stable in human and murine plasma. The stability of the probe was determined by measuring stability in human plasma (>99% remaining) and murine plasma (>89% remaining) after a 5-hour incubation period at 37 °C (Table 2). ML406 showed that it was 99% bound in human plasma and 82.6% bound in mouse plasma (Table 2). The encouraging outcome from this broad spectrum of stability experiments makes ML406 a valuable probe molecule.
Table 2
Plasma Stability and Plasma Protein Binding of ML406 and CID 72836829.
The probe (ML406) and analogue CID 72836829 were found to be stable to both human and murine liver microsomes after 1 hour (Table 3). The stability of ML406 in mouse liver microsomes may provide the opportunity to test this compound in an in vivo mouse model of TB infection (after determining in vitro ADME/PK properties and dose tolerability) as a proof of concept experiment.
Table 3
Liver Microsome Stability of the Probe (ML406, CID 1245700) and analogue CID 72836829.
2.3. Probe Preparation
Probe ML406 was synthesized by the one-step protocol outlined below in Scheme 1. Amidation reaction between benzo[d][1,3]dioxole-5-carboxylic acid and 4-(4-acetylphenyl)piperazin-1-ium 2,2,2-trifluoroacetate in presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and N-ethyl-N-isopropylpropan-2-amine in dichloromethane resulted the probe 1-(4-(4-(benzo[d][1,3]dioxole-5-carbonyl)piperazin-1-yl)phenyl)ethanone (ML406).
Scheme 1
Synthesis of the Probe ML406.
3. Results
3.1. Dose Response Curves for Probe
Figure 2. We identified CID 1245700 from HTS. After several rounds of SAR, the original hit was still found to be optimal and we nominated it as the probe. The re-synthesized sample of the original HTS hit (SID 164225891) also demonstrated similar potencies and the dose response curve of the resynthesized sample are shown in Figure 2.
3.2. Cellular Activity
All growth inhibition assays described in this report are cell-based experiments that measure the activity of compounds against various M. tuberculosis strains. Cytotoxicity was measured against eukaryotic host cells including HEK293, HepG2 and A549 cell lines. A summary of the assays is described in Section 2.1.
3.3. Profiling Assays
No profiling assays were conducted.
4. Discussion
4.1. Comparison to Existing Art and How the New Probe is an Improvement
Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, ISI Web of Science, PubChem, PubMed, US Patent and Trademark Office (USPTO) PatFT and AppFT, and World Intellectual Property Organization (WIPO) databases. Abstracts were obtained for all references returned including journal articles, patents and other form of public disclosures and were analyzed for relevance to the current project. The searches were performed on December 3, 2013, and the results are current as of that date.
The literature and patent searches above identified natural product amiclenomycin as a mechanism-based irreversible inhibitor of the enzyme BioA from Mycobacterium tuberculosis (Mtb) (Figure 3).1-6 The chemical instability and inaccessibility of amiclenomycin prevent it from serving as a useful tool compound.
Figure 3
Amiclenomycin.
A preliminary validation screen with the LOPAC1280 library conducted in the laboratory of Professor Aldrich (Assay Provider for this project) identified six BioA inhibitors (Figure 4).7 CHM-1 was the most potent among these inhibitors (IC50=0.44μM, MIC50=5μM in Mtb under expressing BioA and MIC50>200μM in Mtb over expressing BioA). Follow up studies with CHM-1 revealed that, in the absence of biotin, modest activity (MIC50=50 μM) against wild-type Mycobacterium tuberculosis H37Rv was observed.
Figure 4
Prior Art compounds.
At this point, ML406 shows superior activity profile across all assays over CHM1.
5. References
- 1.
- Okami Y, Kitahara T, Hamada M, Naganawa H, Kondo S. Studies on a new amino acid antibiotic, amiclinomycin. J. Antibiot. 1974;27:656–664. [PubMed: 4436150]
- 2.
- Hotta K, Kitahara T, Okami Y. Studies of the mode of action of amiclinomycin. J. Antibiot. 1975;28:222–228. [PubMed: 805119]
- 3.
- Poetsch M, Zahner H, Werner RG, Kern A, Jung G. Metabolic products from microorganisms. 230. Amiclinomycin-peptides, new antimetabolites of biotin. J. Antibiot. 1985;38:312–320. [PubMed: 3891702]
- 4.
- Sandmark J, Mann S, Marquet A, Schneider G. Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin. J. Biol. Chem. 2002;277:43352–43358. [PubMed: 12218056]
- 5.
- Mann S, Florentin D, Lesage D, Drujon T, Ploux O, Marquet A. Inhibition of Diamino Pelargonic Acid Aminotransferase, an Enzyme of the Biotin Biosynthetic Pathway, by Amiclenomycin: A Mechanistic Study. Helv. Chim. Acta. 2003;86:3836–3850. [CrossRef]
- 6.
- Kitahara T, Hotta K, Yoshida M, Okami Y. Biological Studies of amiclinomycin. J. Antibiot. 1975;28:215–221. [PubMed: 805118]
- 7.
- Wilson DJ, Shi C, Duckworth BP, Muretta JM, Sham YY, Thomas DD, Aldrich CC. A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis. Anal. Biochem. 2011;416:27–38. [PMC free article: PMC3135573] [PubMed: 21621502]
- PMCPubMed Central citations
- PubChem SubstanceRelated PubChem Substances
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- Evaluating the sensitivity of Mycobacterium tuberculosis to biotin deprivation using regulated gene expression.[PLoS Pathog. 2011]Evaluating the sensitivity of Mycobacterium tuberculosis to biotin deprivation using regulated gene expression.Woong Park S, Klotzsche M, Wilson DJ, Boshoff HI, Eoh H, Manjunatha U, Blumenthal A, Rhee K, Barry CE 3rd, Aldrich CC, et al. PLoS Pathog. 2011 Sep; 7(9):e1002264. Epub 2011 Sep 29.
- Structure-Based Optimization of Pyridoxal 5'-Phosphate-Dependent Transaminase Enzyme (BioA) Inhibitors that Target Biotin Biosynthesis in Mycobacterium tuberculosis.[J Med Chem. 2017]Structure-Based Optimization of Pyridoxal 5'-Phosphate-Dependent Transaminase Enzyme (BioA) Inhibitors that Target Biotin Biosynthesis in Mycobacterium tuberculosis.Liu F, Dawadi S, Maize KM, Dai R, Park SW, Schnappinger D, Finzel BC, Aldrich CC. J Med Chem. 2017 Jul 13; 60(13):5507-5520. Epub 2017 Jun 22.
- Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.[Chem Biol. 2015]Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.Park SW, Casalena DE, Wilson DJ, Dai R, Nag PP, Liu F, Boyce JP, Bittker JA, Schreiber SL, Finzel BC, et al. Chem Biol. 2015 Jan 22; 22(1):76-86. Epub 2014 Dec 31.
- Review [Development of antituberculous drugs: current status and future prospects].[Kekkaku. 2006]Review [Development of antituberculous drugs: current status and future prospects].Tomioka H, Namba K. Kekkaku. 2006 Dec; 81(12):753-74.
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