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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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Inhibitors of Protein Folding: DnaK

, , , , , and .

Burnham Institute for Medical Research

Received: ; Last Update: September 2, 2010.

The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family. Specifically, these probes would alter DnaK through interactions with its substrate-binding domain (SBD). DnaK serves as a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression. The identified probe ML076 (CID-25105719) binds to DnaK at a novel allosteric site of the substrate binding domain, in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. ML076 and its closely related analogs described in this probe report might be useful as tools for dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. These molecular probes also might serve to validate DnaK as a possible target for novel antibacterial agents.

Assigned Assay Grant #: X01 MH078942-01

Screening Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Chemistry Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Assay Submitter & Institution: Dr. Maurizio Pellecchia & Sanford-Burnham Medical Research Institute (formerly Burnham Institute for Medical Research)

PubChem Summary Bioassay Identifier (AID): AID-1501

Probe Structure & Characteristics

Chemical name: N-(1-benzothiophen-7-ylmethyl) thiophene-2-carboxamide

Reference name: BI-88D10

MLS: MLS0315930

SID: SID-56427267

CID: CID-25105719

Supplier: Internal BCCG Synthesis

Image ml076fu1
CID/MLTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s )IC50/EC5 0 (μM) [SID, AID]SelectivitySecondary Assay(s)
Name: IC50/EC50 (nM) [SID, AID]

<200,000 nM) [SID-56427267, AID-1033]

Significantly perturbs chemical shift > 0.08 ppm
N/AN/AN/AITC Binding, 200 nM (Kd) [SID-56427267, AID-1495]
ATPase, >20,000,000 nM [SID-56427267, AID-1494]

Recommendations for the scientific use of this probe

The described probe CID-25105719, binds to DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family, at a novel allosteric site of the substrate binding domain in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. The reported compound and its closely related analogs described in this probe report may be useful as tools in dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. In principle, the compounds can find applications also in validating DnaK as possible target for novel antibacterial agents.

1. Scientific Rationale for Project

The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK by specifically interacting with its substrate-binding domain (SBD). DnaK is a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression (Schiene-Fischer 2002, Bukau 2006, Young 2004). The central hypothesis is based on recent observations with some antimicrobial peptides from insects that bind to the SBD of DnaK and that consequently have demonstrated activity against clinical bacterial strains, including fluoroquinoline (Otvos 2000) resistant ones. At least one peptide did not bind to Hsp70 suggesting that it may be feasible to identify selective small molecules inhibitors of DnaK with antimicrobial activity targeting its SBD. DnaK is also very interesting from a protein structure function perspective. It comprises two structural domains (Figure 1, A and B), an N-terminal nucleotide binding ATPase domain (NBD) and a C-terminal substrate binding domain (SBD) which contains a β-strand (β-domain) region that binds the protein substrate and an α-helical lid region that closes over bound substrate (Swain 2007, Chang 2008). DnaK cycles between two main states: an ATP bound state with low affinity for substrate, that is characterized by a tight interaction of the two domains, and an ADP bound state that has high substrate affinity with less interaction between the domains (Hu 2006, Szabo 1994). Structural and biochemical data have shown that residues from both domains (including loop L2,3 of the β-domain), as well as from the linker between them are required for communicating the nucleotide or substrate occupancy to the other respective domain (Swain 2007, Vogel 2006a,b).

With the experience of protein kinase inhibitors in mind (Orchard 2002), screens for Hsp70 inhibitors have focused on compounds targeting the ATP binding site, however, the ATP binding site of Hsp70 is rather hydrophilic and most interaction energy between the nucleotide (ATP or ADP) and the protein is derived from the phosphate groups (Liu 2007). Hence, traditional HTS approaches targeting the ATP binding pocket of Hsp70 or DnaK are unlikely to produce viable hits. Therefore, the proposed NMR-based screen takes the novel approach of directly targeting the substrate binding domain of DnaK. Binders are subsequently used to interrogate in biophysical, biochemical and cell-based assay, the effect of the small molecule on any functional and/or phenotypical parameters.

2. Project Description

a. The original goal for probe characteristics

b. Assay implementation and screening

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

The primary assay was an NMR-based chemical shift perturbation assay (AID-1033) Spectra were acquired on a 600 MHz Bruker Avance equipped with TCI cryoprobe. Ligand binding was monitored by comparing the aliphatic region of 1D 13C-filtered 1H NMR spectra of 20 μM DnaK β-domain only protein solution (20 mM sodium phosphate buffer at pH 7.5 containing 90%/10% H2O/D2O or 99.5% D2O; T= 300 K) in the presence or absence of 80 μM mixtures of 10 compounds, and then individual compound mixtures that caused significant perturbations in the spectrum were characterized further. Simple 1D 1H NMR experiments of the protein measured in presence and absence of mixtures of potential ligands, observing the aliphatic region of the spectra δ<1 ppm). Compound or mixtures causing spectral perturbation above a threshold of 0.08 ppm were deconvoluted.

ii. Assay Rationale & Description

AssayPrimary or SecondaryReagentSource
1D NMR screenPrimaryDnaK β-domain (residues 393–507) w/(MGSSHHHHHHGLVPRGS) at the N-terminusAssay provider Expressed in E. coli BL21(DE3) pLysS Purified by Ni2+ affinity column
2D NMRSecondaryDnaK β-domain 15N, 13C labeled
ITCSecondaryDnaK full - lengthStressgen

The first secondary assay was to confirm binding by NMR by obtaining Kd’s using isothermal calorimetry (ITC; AID-1495). Titrations were done using a VP-ITC from MicroCal (Northampton, MA). Full-length DnaK or SBD were used at 100 μM in 20 mM sodium phosphate buffer (pH 7.4) and 0.5–5% DMSO. Compounds were used at 10–15x in the same buffer.

The ATPase activity (AID-1494) of confirmed binders were determined using the ADAPTA™ kit from Invitrogen at 200 µM of compounds. Positive inhibition would suggest allosteric cross-talk between the domains of DnaK even though compounds bind to only the SBD. This assay cannot be considered a bona fide secondary assay because the other co-chaperone proteins are involved in the process and substrate and cofactor binding and release.

iii. Summary of Results

Using a construct of DnaK corresponding to the β-domain only (residues 393–507) whose solution structures with and without bound peptide are known (Pellecchia 2000, Stevens 2003) a simple 1D 1H NMR based screen of an assembled a scaffold library composed of ~4,000 member was completed by monitoring the aliphatic region of the spectra δ<1 ppm) in the presence and absence pools of 10 compounds at 80 µM per compound binding to 10–20 µM of protein. Mixtures causing spectral perturbation above the threshold of 0.08 ppm were deconvoluted, and additionally reconfirmed for binding by ITC. The compound BI-88B12 emerged as a hit from the screen (Table I, in appendices).

After identifying initial binders, chemical shift mapping studies with 13C and 15N labeled protein suggested that these compounds do not bind to the substrate binding pocket but rather to a pocket located around loop L2,3 on the opposite side of the SBD (Figure 1, A and B). Further molecular docking studies predict binding of compounds BI-88B12 and BI-88D7 in a deep pocket near residues Leu484 and Pro419 reported to be essential for the allosteric communication between the substrate binding and the ATPase domains (Swain 2007, Vogel 2006a,b). These studies also suggested these two compounds bind in different orientations. BI-88B12 places its thiophene ring outside the pocket, this moiety is inserted into the pocket in BI-88D7. For BI-88B12, the fluorobenzene group is inserted deep into the hydrophobic pocket as evidenced by the strongest perturbation of residues there. With its thiophene group in the pocket, the indole ring of BI-88D7 is also able to interact with Asp481, located further out near the edge of the pocket. BI-88D7 causes equal perturbation of both Leu484 and Asp481, possibly due to its increased size that allows it to bridge both residues. This result possibly explains the much lower Kd of BI-88D7 as determined by ITC (see Table 1).

Interestingly, BI-88B12 was indeed found to be an inhibitor of the DnaK ATPase activity with an IC50 of 2 mM, whereas BI-88D7 did not give significant ATPase activity even at 20 mM (Table, below). Interestingly, further studies suggest that the binding of probe BI88D10 alters allosterically the affinity for ATP by ITC (please refer to table 2 of the attached pre-publication manuscript from the assay provider.

c. Probe Optimization

i. SAR & chemistry strategy that led to the probe

An additional 5 commercially available BI-88B12 analogs were qualitatively assessed for their ability to bind the β-domain of DnaK by the magnitude of the chemical shifts induced upon complexation on the 13Cδ,1Hδ resonances of Leu484 (1:10 protein to ligand ratio, at 70 μM protein concentration) by 2D [13C, 1H] correlation spectra. Hit compounds with estimated Kd values of 200 μM or less were selected for further SAR studies including Kd determination by ITC. These data were further analyzed to design 10 additional analogues that were for chemical syntheses. The various ID numbers, structures, molecular weights, activity data and source (commercial or synthesized) for the selected compounds are summarized in Table 1 below:

Table 1Selected purchased & synthesized compounds for SAR Development of confirmed hit BI-88B12

BI88CID [SID]MLS – #StructureMWATPase IC50 (mM)ITC Kd (μM)Source
B12 Initial Hit1244199 [56427257]0014807
Image ml076fu2.jpg
235264.1 ± 8.7 (SBD)ChemBridge
E3743472 [56427258]0100672
Image ml076fu3.jpg
260>2025 (SBD) 4.9ChemBridge
D72814135 [56427263]0311872
Image ml076fu4.jpg
242>20(SBD) 12.7 (FL)Internally Synthesized
D10 Probe25105719 [56427267]0315930
Image ml076fu5.jpg
273>200.2 (FL)Internally Synthesized
D525105722 [56427270]0315933
Image ml076fu6.jpg
267>201.0 (SBD)
1.7 (FL)
Internally Synthesized
D925105724 [56427272]0315935
Image ml076fu7.jpg
255>201.5 (SBD)Internally Synthesized
C51246750 [56427262]0315926
Image ml076fu8.jpg
2960.2–214.3 (SBD)
39.3 (FL)

Of the commercial analogs obtained, BI-88E3 (containing a furan moiety) improved the potency of binding to the SBD of DnaK, but with a loss of ATPase activity. However, BI-88C5 uncovered a compound with improved binding affinity to the DnaK SBD and showed binding to the full-length DnaK, while also recovering a significant amount of ATPase activity, and suggested that substitution of the furan by a thiophene may be a good strategy to improve potency and ATPase activity, though the previous docking studies indicated that despite a shared thiophene ring compounds could bind in different orientations with respect to this thiophene.

Of the ten additional synthesized compounds, significant improvement in affinity to DnaK of about 10-fold was achieved with compounds BI-88D5 and BI-88D9 over the original hit BI-88B12. The most dramatic improvement (>200-fold) was achieved with BI-88D10, though with loss of significant ATPase activity.

3. Probe

a. Chemical name

N-(1-benzothiophen-7-ylmethyl)thiophene-2-carboxamide [ML076]

b. Probe chemical structure

Image ml076fu1

c. Structural Verification Information of probe SID

1H NMR (CDCl3, 600MHz): δ 8.35 (s, 1H), 7.82 (m, 5H), 7.47 (s, 1H), 6.97 (s, 1H), 5.30 (s, 2H); HRESI-TOF-MS: calcd for C14H11NOS2 274.0355 [M + H]+, found 274.0361.

d. PubChem CID (corresponding to the SID)


e. Availability from a vendor

Probe was synthesized internally. A 15 mg sample of the probe compound and 5 analogs have been submitted to the MLSMR.

f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection

Table 2Probe and analog submission

Probe/AnalogMLS-# (BCCG#)CIDSIDSource (vendor or BCCG syn)Amt (mg)Date ordered/submitted
Probe (D10)03159302510571956427267BCCG syn157/9/09
Analog 1 (B12)0014807124419956427257ChemBridge157/9/09
Analog 2 (D7)0311872281413556427263BCCG syn157/9/09
Analog 3 (D5)03159332510572256427270BCCG syn157/9/09
Analog 4 (D9)03159352510572456427272BCCG syn157/9/09
Analog 5 (C5)0315926124675056427262ChemBridge157/9/09

g. Mode of action for biological activity of probe

By attempting to target the substrate binding β-domain of DnaK we have discovered compounds that bind not in the substrate-binding pocket but rather in a cavity involved in allosteric communication between protein domains. We also have discovered compounds with sub-micromolar affinity for DnaK, the most potent to date is the probe molecule CID25105719, as demonstrated by NMR and ITC against the β-domain. Several related analogs also bind the β-domain and the full length protein with single digit micromolar potency. The assay provider intends to use the probe molecule and analogs to “probe” the allosteric communication between domains in vitro and eventually the role of DnaK on bacterial growth in cell and in vivo.

h. Detailed synthetic pathway for making probe

Chemical Synthesis- The actual probe molecule BI-88D10 CID-25105719 [SID-56427267] was synthesized.

The details for synthesized analogs in Table 1 are described in Appendix 4a reported below and the attached pre-print manuscript (see appendix 4c. and Table I below). The QC data for the probe molecule are also listed therein.

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

j. Properties Computed from Structure

Molecular Weight273.37324 [g/mol]
Molecular FormulaC14H11NOS2
H-Bond Donor1
H-Bond Acceptor1
Rotatable Bond Count3
Tautomer Count2
Exact Mass273.028205
MonoIsotopic Mass273.028205
Topological Polar Surface Area85.6
Heavy Atom Count18
Formal Charge0
Isotope Atom Count0
Defined Atom StereoCenter Count0
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count1

4. Appendices

a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes

See Table 1.

The general Scheme for synthesis (Scheme 1) and the detailed synthetic pathway for making selected analogs of Table 1 and 2 are described below.

Scheme 1. General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives.

Scheme 1

General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives.

Chemical Synthesis

To a stirred solution of free amine (1.0 equiv.) and Et3N (2.0 equiv.) in DCM was added a solution of thiophene-2-carbonyl chloride (1.1 equiv.) in DCM at −30 °C. The resulting solution was stirred for 1h and then allowed to warm up to room temperature. After removal of the solvent, the residue was purified by flash column chromatography to provide the correspondent product (yield 75–95%).

Probe molecule (CID-25105719)

BI-88D10-N-(benzo[b]thiophen-7-ylmethyl)thiophene-2-carboxamide 1H NMR (CDCl3, 600MHz): δ 8.35 (s, 1H), 7.82 (m, 5H), 7.47 (s, 1H), 6.97 (s, 1H), 5.30 (s, 2H); HRESI-TOF-MS: calcd for C14H11NOS2 274.0355 [M + H]+, found 274.0361.

BI-88D5- N-(naphthalen-1-ylmethyl)thiophene-2-carboxamide 1H NMR (CDCl3, 600MHz): δ 8.17 (s, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.46 (m, 6H), 6.99 (s, 1 H), 6.39 (s, 1H), 5.01 (s, 2H); HRESI-TOF-MS: calcd for C16H13NOS 268.0791 [M + H]+, found 268.0796.

BI-88D7- N-(1H-indol-5-yl)thiophene-2-carboxamide 1H NMR (CDCl3, 600MHz): δ 8.19 (s, 1H), 7.92 (s, 1H), 7.71 (s, 1H), 7.62 (s, 1H), 7.52 (s, 1H), 7.33 (m, 2H), 7.23 (s, 1H), 7.12 (s, 1H), 6.54 (s, 1H); HRESI-TOF-MS: calcd for C13H10N2OS 243.0587 [M + H]+, found 243.0592. BI-88D6- Is an isomer and side product of BI-88D7 synthesis.

BI-88D9- N-(quinazolin-2-yl)thiophene-2-carboxamide 1H NMR (CDCl3, 600MHz): δ 9.35 (s, 1H), 7.90 (m, 3H), 7.61 (m, 4H), 6.89 (s, 1H); HRESI-TOF-MS: calcd for C13H9N3OS 256.0539 [M + H]+, found 256.0545.

b. Comparative data showing probe specificity for target

See Table 2.

c. Additional information

The assay provider has completed more detailed studies that have been summarized in this probe report. They have also completed additional hypothesis generating experiments that use the probe and some of it’s related analogs as tools to dissect the relative contributions of small molecule binding, ATP nucleotide occupancy, and peptide substrate binding on the binding and function of the full length DnaK protein, that are beyond the scope of the initial MLSCN probe uncover. These do provide a good illustration of the use of small molecule probes on exploring complex interactions related to the specific mechanism of protein allostery for DnaK, and of protein allostery in general.

5. Bibliography

  1. Bukau B, Weissman J, Horwich A. Cell . 2006;125:443–451. [PubMed: 16678092]
  2. Chang YW, Sun YJ, Wang C, Hsiao CD. J Biol Chem . 2008;283:15502–15511. [PMC free article: PMC3258884] [PubMed: 18400763]
  3. Hu B, Mayer MP, Tomita M. Biophys J . 2006;91:496–507. [PMC free article: PMC1483108] [PubMed: 16648174]
  4. Liu Q, Hendrickson WA. Cell. 2007;131:106–120. [PMC free article: PMC2041797] [PubMed: 17923091]
  5. Orchard S. Curr Opin Drug Discov Devel. 2002;5:713–717. [PubMed: 12630291]
  6. Otvos LOI Jr, Rogers ME, Consolvo PJ, Condie BA, Lovas S, Bulet P, Blaszczyk-Thurin M. Biochemistry . 2000;39:14150–14159. [PubMed: 11087363]
  7. Pellecchia M, Montgomery DL, Stevens SY, Vander Kooi CW, Feng HP, Gierasch LM, Zuiderweg ER. Nat Struct Biol. 2000;7:298–303. [PubMed: 10742174]
  8. Schiene-Fischer C, Habazettl J, Schmid FX, Fischer G. Nat Struct Biol. 2002;9:419–424. [PubMed: 12021775]
  9. Stevens SY, Cai S, Pellecchia M, Zuiderweg ER. Protein Sci. 2003;12:2588–2596. [PMC free article: PMC2366956] [PubMed: 14573869]
  10. Swain JF, Dinler G, Sivendran R, Montgomery DL, Stotz M, Gierasch LM. Mol Cell. 2007;26:27–39. [PMC free article: PMC1894942] [PubMed: 17434124]
  11. Szabo A, Langer T, Schroder H, Flanagan J, Bukau B, Hartl FU. Proc Natl Acad Sci U S A. 1994;91:10345–10349. [PMC free article: PMC45016] [PubMed: 7937953]
  12. Vogel M, Bukau B, Mayer MP. Mol Cell. 2006;21:359–367. [PubMed: 16455491]
  13. Vogel M, Mayer MP, Bukau B. J Biol Chem. 2006;281:38705–38711. [PubMed: 17052976]
  14. Young JC, Agashe VR, Siegers K, Hartl FU. Nat Rev Mol Cell Biol. 2004;5:781–791. [PubMed: 15459659]
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