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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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ML328: A Novel Dual Inhibitor of Bacterial AddAB and RecBCD Helicase-nuclease DNA Repair Enzymes

, , , , , , , , , , , , and .

Author Information and Affiliations

Received: ; Last Update: April 5, 2013.

The AddAB and RecBCD helicase-nucleases are related bacterial enzyme complexes that are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although they have been extensively studied both genetically and biochemically, no inhibitors specific for this class of enzymes are known. We developed a high-throughput screen based on the ability of phage T4 gene 2 mutants to grow in E. coli only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized the assay for a 1,536-well plate format and screened the NIH molecular libraries sample collection (326,100 small molecules) for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli RecBCD deletion strain. Two main inhibitor chemotypes, nitrofuran amides and pipemidic acid thioureas, emerged from this cell-based screening campaign, with half maximal effective concentration (EC50) values ranging from 2.5–50 μM. The ultra-high throughput screening (uHTS) campaign was followed by medicinal chemistry structure activity relationship (SAR) optimization, biochemical secondary cell-based and cell-free screening efforts, and mechanism of action studies. These coordinated efforts resulted in the identification of ML328 as a molecular probe. ML328, a drug-like pipemidic acid thiourea, has dual AddAB/RecBCD activity in multiple cell-based and biochemical assays and should prove useful in further enzymatic, genetic, and physiological studies of these enzymes. Because ML328 is a first-in-class inhibitor—no significant prior art exists for potent and selective small molecule inhibition of bacterial AddAB and RecBCD helicase-nucleases—study of this molecular probe could lead to a novel class of drugs to combat infections, since this class of enzymes is widely distributed in bacteria but absent in eukaryotes and is necessary for successful bacterial infection of mammals.

Assigned Assay Grant #: R03 AI 083736

Screening Center Name & PI: The Scripps Research Institute Molecular Screening Center (SRIMSC), H. Rosen

Chemistry Center Name & PI: SRIMSC, H. Rosen

Assay Submitter & Institution: G. Smith, Fred Hutchinson Cancer Research Center

PubChem Summary Bioassay Identifier (AID): 449731

Resulting Publications

A communication describing the screening effort and the uHTS results has been published in 2012 in the journal ACS Chemical Biology. Further publications concerning the activity of ML328 are planned.

1.
Amundsen SK, Spicer T, Karabulut AC, Londono LM, Eberhart C, Fernandez Vega V, Bannister TD, Hodder P, Smith GR. Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes. ACS Chem Biol. 2012;7(5):879–91. [PMC free article: PMC3356449] [PubMed: 22443934]

Probe Structure & Characteristics

ML328.

ML328

CID 1517823

SID 144186606

SR-03000002337-2

molecular weight = 506.5

cLogP = 3.3, cLogD7.4 = 1.6

H-bond acceptors, donors = 8, 2

CID/ML#Target NameIC50 or EC50 [SID, AID]Anti-target NameIC50 [SID, AID]Fold SelectiveSecondary Assay(s): results, [SID, AID]
CID 1517823/ SID 124343048 (purchased)

SID 144186606 (synthesized)
ML328		AddAB and RecBCDAddAB T4 2-assay IC50 = 1.0 μM, [SID 144186606, AID 651942]

AddAB nuclease IC50 = 15.7 μM [SID 12434308, AID 602421]

RecBCD nuclease IC50 = 4.8 μM, [SID 124343048, AID 623920]

RecBCD (Chi cutting) EC50 = 0.6 μM, [SID 124343048, AID 623942]

RecBCD Hfr Rec. V66 EC50 = 0.1 μM, [SID 124343048, AID 623918]		E. Coli V66 Viability34.4 μM
V66 counterscreen [SID 144186606, AID 651943]		34 fold

(comparing V66 assay IC50 vs. AddAB T4 2-assay IC50)		8% remaining AddAB Nuclease Activity at 100 μM, Active [SID 124343048, AID 602422]

8% remaining RecBCD Nuclease Activity at 50 μM, Active [SID 124343048, AID 623922]

−2% remaining RecBCD Nuclease Activity at 100 μM, Active [SID 124343048, AID 623921]

RecBCD Chi Cutting Reduction >2-fold, Active [SID 124343048, AID 623937]

Hfr Recombination V66 Reduction = 6-fold, Active [SID 124343048, AID 623919]

E.coli V66 Hfr cross, 100% Viability at 100 μM, Inactive [SID 124343048, AID 623916]

RecBCD Nuclease IC50, = 4 μM, Active [SID 124343048, AID 623920]

Batch data for CID 1517823

CIDSIDScripps Sample IDSourcePurity
1517823144186606SR-03000002337-2Scripps synthesis98%, (HPLC & NMR)
1517823124343048SR-03000002337-1Vitas-M Lab STK33389495% (HPLC)
Figure 1. Selected properties of probe ML328.

Figure 1Selected properties of probe ML328

1. Recommendations for Scientific Use of the Probe

As an inhibitor of bacterial AddAB and RecBCD helicase-nucleases, ML328 will be immediately useful in understanding how these enzymes help repair damage to bacterial DNA. ML328 has good chemical and liver microsome stability, significantly high water solubility, is not reactive with excess glutathione, is not a significant inhibitor of CYP450 enzymes, and is highly cell permeable. The probe ML328, and any improved derivatives that emerge, will help determine if the inhibition of AddAB and RecBCD helicase-nucleases can prevent bacterial infection or colonization in eukaryotes, which lack these enzymes. In particular, helicase-nuclease inhibitors such as ML328 will be used to study chemoprotection from Helicobacter pylori infection in vivo. Because ML328 is a first-in-class inhibitor—no significant prior art exists for potent and selective small molecule inhibition of bacterial AddAB and RecBCD—its study could lead to a novel class of effective drugs to combat infections.

Introduction

AddAB and RecBCD are related helicase-nuclease bacterial enzyme complexes that are important for the repair of broken DNA and for genetic recombination1–3. Starting at a double-strand (ds) DNA end, these helicase-nucleases unwind DNA rapidly and highly processively, while hydrolyzing ATP or another nucleoside triphosphate. During unwinding they also hydrolyze DNA, making endonucleolytic scissions at a rate dependent on the ratio of [ATP] to [Mg2+], both of which are required for the helicase and nuclease activities. The RecBCD enzyme of E. coli makes endonucleolytic scissions at especially high frequency at Chi sites (5′ GCTGGTGG 3′), which as a consequence are hotspots of recombination4. The RecBCD and AddAB enzymes from other species similarly act at other short nucleotide sequences5. The single-stranded (ss) DNA resulting from unwinding is a potent substrate for the enzymes’ ATP-dependent ss nuclease, which, at least for the RecBCD enzyme of E. coli, produces a digest of primarily tetra- to hexanucleotides6.

Because the helicase-nuclease class of enzymes is important for the repair of DNA double-strand (ds) breaks, mutants lacking them are not viable in mammalian hosts, likely because upon infection the host cells produce reactive oxygen species such as hydrogen peroxide, breaking the bacterial DNA7,8.This finding strongly suggests that small molecule inhibitors of these enzymes will be effective as antibacterial drugs. AddAB and RecBCD are widely distributed among both gram-negative and gram-positive bacteria, indicating potential for broad utility of effective inhibitors of these enzymes.

Potent and specific small molecule enzyme inhibitors are exceptionally useful tools in bacterial enzymology and physiology, especially for elucidating pathways and defining mechanisms whereby mammals may be protected from unwanted bacterial infection. Small molecule probes can be particularly useful in the study of multifunctional enzymes, such as the helicase-nucleases, where they can halt a multi-step enzyme-driven process at an intermediate stage, allowing for the identification of reaction intermediates and thus permitting a deeper mechanistic understanding of the pathway. Such probes are essential for spurring drug discovery efforts. Notable examples include the aminocoumarin and quinolone classes of DNA gyrase inhibitors and the camptothecin class of topoisomerase I inhibitors. In these cases, early inhibitors were valuable tools for understanding how the protein targets interact their DNA substrates9,10. As an example, such mechanistic insights aided the optimization of quinolone inhibitors, a class of drugs that is particularly effective because of the prevalence of DNA gyrase in bacteria but not in eukaryotes. Quinolones such as ciprofloxacin are commercially successful front-line antibacterial therapeutics.

Although biochemical and genetic studies of the AddAB and RecBCD enzyme complexes have spanned over 50 years1,11, no functionally specific small molecule inhibitors for these enzymes have been reported. Dziegielewska et al.12 reported RecBCD helicase inhibition by three compounds that function by binding and alkylating DNA in a non-specific manner, strongly suggesting that these compounds will inhibit other helicases and enzymes acting on DNA as well. Inorganic species such as Ca2+ and other divalent ions non-specifically inhibit E. coli RecBCD and B. subtilis AddAB nuclease, though not the helicase13–15. The Gam protein of phage λ also partially inhibits RecBCD16,17, perhaps by binding to the site at which DNA binds18,19. Non-specific inhibitors, such as EDTA and SDS, have also been used to halt RecBCD activity, but by lacking specificity they have little utility for mechanistic studies.

We have sought small molecule drug-like and target-specific inhibitors of RecBCD and AddAB, both to aid mechanistic studies and to serve as a novel class of potentially therapeutically useful antibacterial agents. As a prelude to screening libraries of small molecules, we developed a cell-based assay based on the ability of phage T4 gene 2 mutants to grow in E. coli only if the RecBCD nuclease is inactivated by mutation20. The cell-based format assures that only compounds able to enter E. coli are identified, i.e.; all screening hits have the advantage of significant cell permeability. Nuclease activity resides in the RecB polypeptide21 but it is active only if the RecD subunit is also present, and moreover only if the RecB helicase is also active22,23. The RecB helicase, in turn, is highly active only in the presence of the RecC subunit24. In the AddAB enzyme complex, each subunit contains a nuclease domain; only AddA appears to have an active helicase domain, but its inactivation blocks all detectable nuclease activity13,25–27. Consequently, small molecules that bind to any subunit of either enzyme might inhibit the nuclease, either directly or indirectly, and allow T4 gene 2 mutant phage to grow, thereby blocking the growth of E. coli.

Our uHTS strategy was designed to identify cell-permeable small molecule inhibitors of AddAB and RecBCD. Our uHTS effort was complemented by a focused follow-up SAR development strategy, a coordinated medicinal chemistry and molecular biology effort intended to optimize properties of the active compounds that were identified, to define their mode of action, to show their relevance, and finally to deliver a potent and selective AddAB/RecBCD inhibitor probe. The resulting probe compound, ML328, belongs to the pipemidic acid thiourea scaffold. ML328 is the first described potent small molecule known to selectively modulate the activity of these enzymes. It is a dual inhibitor, with activity vs. both AddAB and RecBCD in cell-based and biochemical assays. It has high efficacy (EC50 ~0.1 μM) seen in certain secondary cell-based assays, such as for RecBCD Hfr recombination. Dual inhibition of AddAB and RecBCD may confer synergistic effects in halting proliferation of organisms such as Mycobacterium spp. that have both enzymes28.

The simplicity of the structure of ML328, its relative ease of preparation, its favorable physical characteristics, and its biological properties all contribute to its potential use as a molecular probe useful for understanding the role(s) of AddAB and RecBCD in bacterial growth and survival.

2. Materials and Methods

Chemistry: All chemical reagents and solvents were acquired from commercial vendors. Reactions were monitored by LC/MS (Thermo/Finnegan LCQ Duo system with MS/MS capability). An Agilent 1200 analytical HPLC was used for quantitative purity assessment. Teledyne-Isco “combiflash” automated silica gel MPLC instruments were used for chromatographic purifications. A 400 Brüker MHz NMR instrument was used for NMR analysis.

Biology: All protocols are reported in the relevant PubChem AIDs, provided below.

Evaluation of compound properties: Solubility, stability, and glutathione reactivity analyses were conducted in accordance with NIH guidelines. CYP450 inhibition and microsome stability analyses were performed as previously described29.

2.1. Assays

Table 1 lists AddAB-related PubChem AIDs. Descriptions of the assays follow the table.

Table 1. AddAB Inhibitors PubChem Summary (Cycle 13).

Table 1

AddAB Inhibitors PubChem Summary (Cycle 13).

Assay Descriptions

Click on the hyperlinked text to see the assay details in PubChem.

AddAB Inhibition T4 2- Assays (AID 435030, AID 488942, AID 492959, AID 504677, and AID 651942)

The purpose of this assay is to identify compounds that act as inhibitors of bacterial AddAB activity. This bacterial cell-based assay employs E. coli that express the Helicobacter pylori addAB genes. The bacteria are infected with a mutant T4 bacteriophage that carries three nonsense mutations in gene 2, whose protein product normally protects viral DNA from AddAB-mediated degradation after infection. The mutant phage infects and blocks the growth of strain V3069 E. coli, which lack AddAB nuclease activity (AddAB-). The mutant phage also infect strain V3065 E. coli, which contain a plasmid expressing the H. pylori addAB+ genes, but V3065 proliferate because of the AddAB directed nuclease and helicase activity against the unprotected mutant phage. Both strains lack E. coli RecBCD (recBCDΔ). In this assay, the V3065 E. coli cells are infected with mutant T4 phage in the presence of test compounds, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit AddAB will allow the virus to replicate and inhibit bacterial growth, leading to reduced well absorbance. Compounds are tested in singlicate (AID 435030) and triplicate (AID 488942) at a nominal test concentration of 11.86 μM, and in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 118.6 μM (AID 492959 and AID 504677: powders Round 1; AID 651942 powders Round 2).

Prior to the start of the assay, V3065 and V3069 bacterial cultures were grown at 37°C until they reached an OD600 of 0.05 or 2.5e07 cfu/mL. To start the assay, 3uL of Assay Buffer (0.1% Glycerol + 100 μg/mL Ampicillin + Cation-adjusted Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 μg/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells. Then, 1 μL of V3065 (addAB+) or V3069 (phage control) bacterial cultures were dispensed into the appropriate wells and plates were incubated for 60 minutes at 37°C. Next, 1 μL of mutant T4 gene 2 mutant 149 mutant bacteriophage was dispensed to the appropriate wells at a multiplicity of infection (MOI) of 0.02. Plates were centrifuged and after 18 hours of incubation at 37°C, absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well. The percent inhibition for each compound was calculated as follows:

% Inhibition = 100 * ((Test_Compound − Median_Low_Control) / (Median_High_Control − Median_Low_Control))

Where:

  • High_Control is defined as wells containing V3065 + Ciprofloxacin + phage.
  • Low_Control is defined as wells containing V3065 + DMSO + phage.
  • Test_Compound is defined as wells containing V3065 in the presence of test cmpd + phage.
RecBCD T4 2- Assays (AID 488955, AID 492957, AID 504679, AID 651944)

The purpose of this assay is to identify compounds identified as active in a set of previous experiments entitled, “Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complex” (AID 435030), that also inhibit E. coli RecBCD, as an early indication of a compound that may have potential to elicit a broad-spectrum antibiotic effect. This assay involves infecting E. coli with a T4 bacteriophage that carries three nonsense mutations in gene 2, whose wild-type protein product protects viral DNA from RecBCD-mediated degradation after infection. The mutant T4 phage is able to infect and block the growth of strain V67 E. coli (recB21, a recBCD null mutation), which lack RecBCD nuclease activity. The mutant phage also infects strain V66 E. coli (recBCD+), but V66 proliferate because of the RecBCD helicase and nuclease activity against the unprotected mutant phage. In this assay, the V66 E.coli cells are infected with mutant T4 phage in the presence of test compounds, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit RecBCD will allow the virus to replicate and block bacterial growth, leading to reduced well absorbance. Compounds are tested in triplicate at a nominal test concentration of 11.86 μM (AID 488955), in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 118.6 μM (AID 492957: liquids, (AID 504679, and AID 651944 powders).

Prior to the start of the assay, V66 and V67 bacterial cultures were grown at 37°C until they reached an OD600 of 0.05 or 2.5e07 cfu/mL. To start the assay, 3 μL of Assay Buffer (0.1% Glycerol + Cation-adjusted Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 μg/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells. Then, 1 μL of V66 (recBCD+) or V67 (phage control) bacterial cultures were dispensed into the appropriate wells and plates were incubated for 60 minutes at 37°C. Next, 1 μL of mutant T4 gene 2 mutant 149 mutant bacteriophage was dispensed to the appropriate wells at a multiplicity of infection (MOI) of 0.02. Plates were centrifuged and after 18 hours of incubation at 37°C, absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well. The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * ((Test_Compound - Median_Low_Control) / (Median_High_Control - Median_Low_Control))

Where:

  • High_Control is defined as wells containing V66 + Ciprofloxacin + phage.
  • Low_Control is defined as wells containing V66 + DMSO + phage.
  • Test_Compound is defined as wells containing V66 in the presence of test compound + phage.
V3065 and V66 Bacterial Viability Counterscreens (AID 449728, AID 488956, AID 492958, and AID 651943)

The purpose of these assays is to identify compounds that are nonselective inhibitors of the AddAB helicase-nuclease complex due to bacterial cytotoxicity. In the V3065 assay, V3065 bacteria are not infected with phage but are incubated in the presence of test compounds alone, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit bacterial growth will reduce well absorbance. Compounds are tested in singlicate (AID 449728) and in triplicate (AID 488956) at a nominal test concentration of 11.86 μM, and in triplicate using a 10-point 1:3 dilution series starting at a nominal test concentration of 118.6 μM (AID 492958: liquids, and AID 651943: powders).

Prior to the start of the assay, V3065 bacterial culture was grown at 37°C until it reached an OD600 of 0.05 or 2.5e07 cfu/mL. V3065 was diluted in assay buffer to achieve 1.25e07 cfu/mL. To start the assay, 3 μL of Assay Buffer (0.1% Glycerol + 100 ug/mL Ampicillin + Cation-adjusted Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 μg/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells. Next, 2 μL of V3065 (AddAB+) bacterial culture was dispensed into the appropriate wells. Plates were incubated for 18 hours at 37°C and absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well. The percent inhibition for each compound was calculated as follows:

100 * ((Test_Compound − Median_Low_Control) / (Median_High_Control − Median_Low_Control))

Where:

  • High_Control is defined as wells containing V3065 + ciprofloxacin.
  • Low_Control is defined as wells containing V3065 + DMSO.
  • Test_Compound is defined as wells containing V3065 in the presence of test compound.

In the V66 variant of this assay, the same procedures are followed except using E. coli. V66. The team determined that this assay is most appropriate to gauge inherent, non nuclease/helicase-related cellular toxicity of probe candidates, since V66 is normal with respect to DNA repair capabilities, whereas strain V3065 is deficient for DNA damage repair.

AddAB Nuclease Assays (AID 602422 and AID 602421)

The purpose of this assay is to determine whether fresh batches (“powder samples”) of newly-prepared compounds previously identified as probe candidates can inhibit AddAB enzymatic activities, using AddAB purified from E. coli. Nuclease activity is assayed as the formation of trichloroacetic acid-soluble radioactive material, detected with a scintillation counter, from uniformly labeled [3H] double-stranded (ds) DNA. As designed, compounds that inhibit AddAB will reduce the nuclease activity (less acid-soluble material after standard incubation). In this assay compounds were tested at a single nominal concentration of 100 μM (AID 602422) and using a dose response series up to a maximum nominal concentration of 100 μM (AID 602421).

Nuclease assays measured the formation of TCA-soluble radioactive material from phage T7 [3H] DNA (2 μg/mL; 6 μM nucleotides) substrate in a 20 min incubation at 37°C. AddAB assays were in 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, polyvinylpyrrolidone (1 mg/mL), 1 mM DTT, and 50 μM ATP. 2 nM AddAB enzyme was used in this assay. Compounds were diluted in DMSO and added to enzyme in assay buffer on ice; final DMSO concentration was 5.0% (v/v) in each assay. DNA substrate was added, and after <5 minutes the reactions were started by bringing the samples to 37°C. Reactions were stopped by addition of calf thymus DNA to 0.2 mg/mL and TCA to 5% (w/v). After 10 minutes on ice, the mixtures were centrifuged for 5 min at 16,100 × g, and the soluble radioactive material was determined in a scintillation counter. The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * (1 - ((Test_Compound - Background) / (Mean_DMSO_Control - Background)))

Where:

  • Mean_DMSO_Control is defined as TCA-soluble radioactive product formed by AddAB ds exonuclease in 20 minutes with the presence of 5.0 %(v/v) DMSO.
  • Background is defined as TCA-soluble radioactive product formed without AddAB enzyme.
  • Test_Compound is defined as TCA-soluble radioactive product formed in the presence of compound diluted in 5.0% (v/v) DMSO.
RecBCD Nuclease Assays (AID 623920, AID 623921 and AID 623922)

The purpose of this counterscreen assay is to determine whether powder samples of compounds identified as AddAB probe candidates can inhibit RecBCD enzymatic activities. To begin to elucidate the mechanism of inhibition, nuclease activity was assayed using RecBCD purified from E. coli. Nuclease activity is assayed as the formation of trichloroacetic acid-soluble radioactive material, detected with a scintillation counter, from uniformly labeled [3H] double-stranded (ds) DNA. As designed, compounds that inhibit RecBCD will reduce the nuclease activity (less acid-soluble material after standard incubation). Compounds are tested in triplicate using a dilution series starting at a nominal maximum test concentration of 100 μM (AID 623920: liquids, AID 623921: powders), and in triplicate at a nominal test concentration of 50 μM (AID 623922: powders).

Nuclease assays measured the formation of TCA-soluble radioactive material from phage T7 [3H] DNA (2 μg/mL; 6 μM nucleotides) substrate in a 20 minute incubation at 37°C. RecBCD assays were in 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, polyvinylpyrrolidone (1 mg/mL), 1 mM DTT, and 25 μM ATP. 0.15 units of RecBCD enzyme per 50 μL reaction (4.5 nM) was used in this assay.

Compounds were diluted in DMSO and added to enzyme in assay buffer on ice; final DMSO concentration was 5.0% (v/v) in each assay. DNA substrate was added, and after <5 min the reactions were started by bringing the samples to 37°C. Reactions were stopped by addition of calf thymus DNA to 0.2 mg/mL and TCA to 5% (w/v). After 10 minutes on ice, the mixtures were centrifuged for 5 min at 16,100 × g, and the soluble radioactive material was determined in a scintillation counter. The percent RecBCD activity following treatment with compounds was calculated as follows:

%_Enzyme_Activity = 100 * (1- ((Test_Compound - Background) / (Mean_DMSO_Control - Background)))

Where:

  • Mean_DMSO_Control is defined as TCA-soluble radioactive product formed by RecBCD ds exonuclease in 20 min in the presence of 5.0 %(v/v) DMSO.
  • Background is defined as TCA-soluble radioactive product formed in the absence of RecBCD enzyme.
  • Test_Compound is defined as TCA-soluble radioactive product formed in the presence of compounds diluted in 5.0% (v/v) DMSO.

For each test compound, percent inhibition was plotted against compound concentration using Prism 5 software (GraphPad Software, Inc). The reported IC50 values were generated in GraphPad. In cases where the highest concentration tested (i.e.; 100 μM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 μM.

RecBCD Chi Cutting Assays (see AID 623937 and AID 623942)

The purpose of this assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit the ability of E. coli RecBCD enzyme to cut DNA at a Chi sequence (5′GCTGGTGG 3′) during DNA unwinding. In this assay linear, 5′ end-labeled duplex DNA with Chi is incubated with RecBCD in the absence and presence of compound, and the products are analyzed by gel electrophoresis and autoradiography. A labeled fragment ending at or near Chi, but absent in reactions with DNA lacking Chi, reflects Chi-specific cutting. Controls include native and boiled DNA without RecBCD. As designed, an inhibitor of RecBCD will decrease the amount of the Chi-specific fragment. If the compound inhibits DNA unwinding, less full length single-stranded DNA, with mobility equal to that of boiled DNA, will be seen. This assay is designed to determine if compounds inhibit either the unwinding of DNA or specific cutting at Chi hotspots of recombination. Compounds were tested at a nominal concentration of 50 μM (AID 623937) and using a dilution series starting at a maximum nominal concentration of 500 μM (AID 623942).

Helicase assays measured the formation of ss DNA from [5′ 32P] pBR322 χ+F (or χo control)4 DNA (0.1 nM molecules) linearized by digestion with Hind III enzyme. RecBCD unwinding assays were in 25 mM Tris-acetate (pH 7.5), 2.0 mM Mg(OAc)2, 5 mM ATP. RecBCD assays used 0.15 nM enzyme and without SSB. Compounds were added to the reaction mixture containing all the reagents except ATP; final DMSO concentration was 2.5% (v/v) for each assay. (All other percentages in this paragraph are w/v.) Reactions were started by addition of ATP and were at 37°C for 1 minute. Reactions were stopped by addition of 1/3 volume of stop buffer (2.5% SDS, 100 mM EDTA, 0.125% bromophenol blue, 0.125% xylene cyanol FF, and 10% Ficoll), and the products were subjected to electrophoresis in a 1.25% agarose gel at 5 V/cm for 2.5 hours in TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA). Gels were dried under vacuum, and the products were detected by autoradiography or analyzed with a Typhoon Trio PhosphorImager (GE Lifesciences) and ImageQuant TL software (Amersham). With RecBCD, this assay also detects cutting of DNA at the Chi site χ+F, which produces a 1.46 kb 5′ labeled fragment. The fold reduction in RecBCD activity for each compound was determined according to the degree of reduction of Chi cutting as observed on the X-Ray film of the dried gels. The activity was determined as follows:

Fold_Reduction = (Test_Compound_Chi_Band_Intensity)/(DMSO_Control_Chi_Band_Intensity)

Where:

  • DMSO_Control_Chi_Band_Intensity is defined as the intensity of the Chi band formed in the presence of 5% DMSO.
  • Test_Compound_Chi_Band_Intensity is defined as the intensity of the Chi band formed in the presence of test compound dissolved in 5% final concentration of DMSO in the assay mixture.
RecBCD Hfr Recombination Assays (see AID 623919 and AID 623918)

The purpose of this assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit recombination in Hfr conjugational crosses. In this assay a test strain is mated with an Hfr strain in the presence of compound, and the frequency of selected recombinants (His+ StrR) in the mixture is determined by differential plating. The donor and recipient are also plated on non-selective medium to measure viability. As designed, compounds that inhibit RecBCD will reduce the frequency of recombinants. This assay is designed to determine if inhibitors of RecBCD can enter living cells and inhibit the recombination-promoting function of RecBCD. E. coli are exposed to test compounds for 1.5 hours. Recipient cells are grown for 1 hour in LB broth with or without compound. Donor and recipient cells are plated separately on non-selective media to measure viability. Donor (Hfr) and recipient (F−) cells are mixed in a ratio of 1:10, incubated for 30 minutes, vortexed to separate mating cells, and plated differentially to determine the frequency of recombinants (His+ StrR). Probes should be active in these assays. Compounds are tested at a nominal concentration of 100 μM in duplicate experiments (AID 623919) and using a 5-point dilution series of doses ranging from 0.03 to 100 μM (AID 623918). The relative viability of each culture was calculated as follows:

Relative_Viability = the number of colony forming units per ml of culture treated with compound normalized to the number of colony forming units per ml of culture treated with DMSO only.

The fold reduction in recombinant frequency for each compound was calculated as follows: Fold_Reduction = [Recombinant frequency of DMSO control] / [Recombinant frequency in presence of compound adjusted for relative viability of the culture].

Compounds that reduced V66 E. coli recombinant frequency by less than or equal to 4-fold compared to the DMSO control were considered inactive. Compounds that reduced V66 E. coli recombinant frequency by greater than 4-fold compared to DMSO control were considered active.

V66 Hfr Conjugational Cross Bacterial Viability Counterscreens (AID 623916 and AID 623917)

The purpose of this assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates reduce the viability of the recipients in Hfr conjugational crosses. In this assay a test strain is grown in the presence of compound for 1 hr, mated with an Hfr strain in the presence of compound, and the frequency of selected recombinants (His+ StrR) in the mixture is determined by differential plating. The viability of the donor and recipient are determined by plating on non-selective media. E. coli cells were exposed to test compounds for 1.5 hours. As designed, compounds that reduce strain V66 viability could reduce the apparent frequency of recombinants. This assay is designed to determine if compounds can enter living cells and reduce viability. As part of the Hfr recombination crosses, compounds were tested at a single nominal concentration of 100 μM (AIDs 623916) and using a 5-point dilution series of doses ranging from 0.03 to 100 μM (AID 623917).

Recipient cells are grown for 1 hour in LB broth with or without compound. Donor and recipient cells are plated on non-selective media to determine viability. The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * (1 - [colony forming units per ml of V66 in presence of compound] / [colony-forming units per ml of V66 in DMSO control])

This value is used to adjust the frequency of His+ StrR recombinants determined in assays of Hfr recombination.

E. coli Recombination Assays in the Presence of AddAB (AID 623956 and AID 623954)

The purpose of this assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit recombination promoted by H. pylori AddAB in Hfr conjugational crosses. In this assay an E. coli recBCD deletion test strain transformed with plasmids bearing H. pylori addAB+ and recA+ is mated with an Hfr strain in the presence of compound, and the frequency of selected recombinants (His+ StrR) in the mixture is determined by differential plating. The viability of the donor and recipient strains are determined by plating on non-selective media. E. coli were exposed to test compounds for 1.5 hours. As designed, compounds that inhibit AddAB will reduce the frequency of recombinants. This assay is designed to determine if inhibitors of AddAB can enter living cells and inhibit the recombination-promoting function of AddAB. Probes should be active in this assay. Compounds were tested in duplicate experiments at single doses of 20 μM (AID 623956) and in duplicate experiments at a single dose of 100 μM (AID 623954).

Recipient cells are grown for 1 hour in LB broth with or without compound. The viability of the donor and recipient strains are determined by plating on non-selective media. Donor (Hfr) and recipient (F−) cells are mixed in a ratio of 1:10, incubated for 30 minutes, vortexed to separate mating cells, and plated differentially to determine the frequency of recombinants (His+ StrR). The relative viability of each culture was calculated as follows: Relative_Viability = [colony forming units per ml of culture with compound] / [colony forming units per ml of culture with DMSO]. The fold reduction in recombinant frequency for each compound was calculated as follows:

Fold_Reduction = [recombinant frequency of DMSO control] / [recombinant frequency in presence of compound adjusted for relative viability of the culture]

Where:

  • Recombinant_Frequency = 100 * [His+ StrR recombinants per donor cell adjusted for the viability of the recipient]

Compounds that reduced recombinant frequency (viability) by 4-fold or greater compared to the DMSO control were considered active. Compounds that reduced recombinant frequency (viability) less than 4-fold were considered inactive.

2.2. Probe Chemical Characterization

The chemical structure of the probe was verified by analysis of its 400 MHz 1H NMR spectra (Figure 2) obtained on a Brüker 400 MHz instrument. The chemical structure was also corroborated by its LC/MS molecular ion (calc for M+1: 507.1, found 507.03, using an Agilent 1200/6140 multimode quadrupole rapid resolve system). Purity was measured at >98% (LC/MS analysis, confirmed by analytical HLPC analysis. The mass spectrum is shown in Figure 3. HPLC purity data is shown in Figure 4. HPLC data was obtained using an Agilent 1200 analytical HPLC with an Agilent Eclipse XDB-C18 column, 4.6×150mm. The HPLC solvents used were acetonitrile and water with 0.1% formic acid added to each mobile phase as the pH modifier.

Figure 2. 1H NMR spectrum.

Figure 2

1H NMR spectrum.

Figure 3. Mass spectrum.

Figure 3

Mass spectrum. Calc for M+1: 507.1, found 507.03.

Figure 4. HPLC spectrum.

Figure 4

HPLC spectrum. Purity >95%.

The solubility of the probe ML328 in PBS at pH 7.4 was determined to be 47 μM. Its solubility is fully adequate to provide the high potency seen in cell-based assays and is also adequate for broad use as a biological probe to be used in a variety of aqueous-based media.

The probe ML328 has a half-life of over 48 hours in PBS at room temperature when tested at 10 μM. Disappearance of the LC peak for the probe is unaltered by the addition of excess glutathione (50 μM) in a 6 hour incubation. The probe is stable in DMSO solution at room temperature (no erosion of peak intensity over 48 hours) and is also stable as a free base dry powder. It is also stable under assay conditions, as indicated by potency in various secondary assays that is independent of incubation time.

The compounds in Table 2 have been submitted to the MLSMR collection.

Table 2. Probe and Analogs.

Table 2

Probe and Analogs.

2.3. Probe Preparation

The probe ML328 was synthesized in a straightforward fashion in a single step using commercially available reagents, as shown in Figure 5. The overall yield was 54%. Analogs for SAR evaluation were prepared by similar methods.

Figure 5. One Step Synthesis of ML328.

Figure 5

One Step Synthesis of ML328.

To an oven dried 100 mL round bottom flask equipped with a magnetic stir bar was added pipemidic acid (0.250 g; 0.824 mmol), 3(trifluoromethyl)phenyl isothiocyanate (0.167 g; 0.823 mmol), and sodium bicarbonate (0.083 g; 0.988 mmol), and the flask was flushed with Argon for 10 minutes. Dry N,N-dimethylformamide was added (40 mL) and the flask was once again flushed with Argon. The reaction mixture was allowed to stir for 15 hours at room temperature. After confirming the completion of reaction by TLC and LC-MS, the reaction was quenched by addition of 25 mL of a saturated NH4Cl solution. The contents were transferred to a separatory funnel and extracted with ethyl acetate (3 × 25 mL). The organic extracts were combined and washed with water (3 × 25 mL), brine (1 × 25 mL), and dried over Na2SO4. Finally the solvents were evaporated on a rotary evaporator to give a crude solid. The crude material was then purified by flash chromatography (10% MeOH in CH2Cl2) to give 0.225 g (54% yield) of ML328, The purity of this compound was confirmed by 1H NMR, MS, and analytical HPLC. 1H NMR (400 MHz, CDCl3): δ = 14.82 (s, 1H), 9.64 (s, 1H), 9.28 (s, 1H), 9.02 (s, 1H), 7.75 (br s, 1H), 7.69 (d, J = 7.6 Hz, 1H). 7.55 (t, J = 7.5 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.12 (m, 8H), 1.39 (t, J = 7.4 Hz, 3H) ppm; LCMS Calc’d for M+1 = C22H22F3N6O3S: 506.14, Found: 507.03.

3. Results

3.1. Dose Response Curves for Probe

AddAB T4 2- assay results. This bacterial cell-based assay employs E. coli that express the Helicobacter pylori addAB+ genes. The bacteria are infected with a mutant T4 bacteriophage that carries three nonsense mutations in gene 2, whose protein product normally protects viral DNA from AddAB-mediated degradation after infection. The assay was used in the uHTS campaign and in follow-up runs to aid probe development AID 435030, AID 488942, AID 492959, AID 504677, AID 651942). The probe compound displayed an EC50 = 1.0 μM in this assay (SID 144186606, AID 651942)

Figure 6. ML328 in AddAB T4 2- assay, SRIMSC.

Figure 6ML328 in AddAB T4 2- assay, SRIMSC

EC50 = 1.0 μM, max % response = 99 Hill slope = 1.32

RecBCD biochemical assay results: nuclease activity. This biochemical assay was routinely run in support of probe development efforts (AID 623920, AID 623921, AID 623922). The probe compound was analyzed several times in this assay, with consistent low μM potency seen each time. Figure 7 is a representative curve (SID 124343048, AID 623920).

Figure 7. RecBCD nuclease assay.

Figure 7

RecBCD nuclease assay. (Assay Provider) IC50 = 4.8 μM

AddAB biochemical assay results: nuclease activity. This biochemical assay was also routinely run in support of probe development efforts (AID 602422 and AID 602421). The probe compound was analyzed several times in this assay, with consistent mid μM potency seen each time. Figure 8 is a representative curve (SID 124343048, AID 602421)

Figure 8. AddAB nuclease assay.

Figure 8

AddAB nuclease assay. (Assay Provider) IC50 = 15.7 μM

We also profiled the probe compound in a variety of secondary assays, including RecBCD Chi cutting and RecBCD Hfr recombination assays, where we obtained EC50 values of 0.6 μM and 0.1 μM, respectively. These results will be discussed in section 3.3 under profiling assays.

V66 Bacterial Viability Counterscreens (anti-target). This assay was used as a marker for non AddAB-specific E. coli cytotoxicity for probe candidates and was a variant of the assay used in the uHTS effort and as needed to support probe development, with strain V3065 (AID 492958, AID 504678). The probe compound gave an IC50 = 34.4 μM in this assay (~35-fold higher than the AddAB T4 2- cell-based result). This low off-target toxicity is also consistent with observations in other cell-based assays where there was no observed effect on cell growth or viability caused by the probe, even at doses over 50 μM.

Figure 9. E. coli V66 viability (anti-target assay), SRIMSC.

Figure 9E. coli V66 viability (anti-target assay), SRIMSC

IC50 = 34.4 μM

3.2. Cellular Activity

The uHTS assays and many follow-up primary and secondary assays are cell-based, so the probe has confirmed cellular permeability.

3.3. Profiling Assays

We recently published a communication describing the screening effort and the uHTS results34. In that communication a great deal of profiling of the screening hit (CID 1045135) was disclosed, including a RecBCD Chi-cutting assay and a cell-based assay for RecBCD-dependent inhibition of intracellular recombination. We were surprised that CID 1045135 showed significant effects at much lower concentrations in these assays (~10X lower), as compared to the biochemical assays using purified enzyme. This result may reflect differences in the enzyme’s environment during the assays, or it may reflect some activity of RecBCD (which is a large [330-kDa] complex of three polypeptides) that we have not yet assayed, such as the loading of RecA protein after action at Chi36, that is even more sensitive to inhibition than the nuclease, DNA unwinding, and Chi-cutting. In any case, this result encourages the possibility that these or related compounds may be effective antibacterial drugs.

The results with the screening hit carried over (with concomitant added potency) when the probe compound, ML328, was evaluated. In the RecBCD Chi cutting assay an IC50 = 0.6 μM was measured [SID 124343048, AID 623942] and significant effects at even lower doses was seen in the RecBCD Hfr Recombination assay, EC50 = 0.1 μM, [SID 124343048, AID 623918].

The pipemidic acid thioureas are structural similar to quinoline antibiotics such as norfloxacin and ciprofloxacin, yet the screening hit (CID 1045135) and analogs are not significantly potent inhibitors of gyrase (data not shown), thus their effects are based upon their helicase-nuclease activity.

As mentioned earlier, the probe has measurable effects on viability of AddAB-deficient E. coli V66 though the selectivity window is ~35-fold, indicating a lack of non-helicase-nuclease associated toxicity.

Analysis of pubchem data shows that the screening hit (CID 1045135) is active in only a small percentage of all assays in which it has been screened (~1.2%, N=499), indicating a lack of non-specific protein binding or polypharmacology, characteristics that likely extend to the probe ML328, a positional isomer of the screening hit. There are no serious issues with regard to the drug-like attributes of the probe30–32. There are also no concerns with respect to toxicity structure alerts33.

AddAB and RecBCD are structurally very similar, both with helicase and nuclease activities that are dependent on ATP. Thus the finding that a class of molecules can inhibit both enzymes was somewhat expected. To compare published structures see PDB 1W36 for E. coli RecBCD and 3U4Q for B. subtilis AddAB. Dual nuclease and helicase activity was also not unexpected, as the nuclease activity of RecBCD and AddAB depends on the helicase activity, which in turn depends on the ATPase activity. We surmise that the compounds directly inhibit the helicase or the ATPase activity and as a consequence inhibit the nuclease activity, including cutting at Chi sites. We intend to determine the mode of action in future studies.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

As discussed in the introduction, no functionally specific small molecule inhibitors for the bacterial AddAB and RecBCD enzyme complexes have been reported. Dziegielewska et al.12 reported RecBCD helicase inhibition by three compounds that function by binding and alkylating DNA in a non-specific manner, strongly suggesting that these compounds will inhibit other helicases and DNA enzymes as well. Inorganic species such as Ca2+ and other divalent ions non-specifically inhibit E. coli RecBCD and B. subtilis AddAB nuclease, though not the helicase13,14,15. The Gam protein of phage λ also partially inhibits RecBCD16,17, perhaps by binding to the site at which DNA binds18,19. Non-specific inhibitors, such as EDTA and SDS, have also been used to halt RecBCD activity, but by lacking specificity they have little utility for mechanistic studies.

The lack of any relevant prior art is a key strong point of the probe ML328. Moreover, ML328 possesses many desirable properties (potency, solubility, lack of toxicity, chemical stability, liver microsome stability, lack of CYP450 inhibition, favorable drug-likeness) that should aid its use.

5. References

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6

Present address: Kineta, Inc., 219 Terry Avenue North, Suite 300, Seattle, Washington 98109-5208;

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