2.1. Assays

Details for the assays listed in Table 3 are provided in Appendix A.

Table 3

List of Assays and AIDs for this Project.

2.2. Probe Chemical Characterization

Probe Chemical Structure, Physical Parameters, and Probe Properties

Figure 1Property summary of probe compound ML283/SID 114279600 (CID 50930730)

Stability

The aqueous stability of ML283 was measured with and without the addition of acetonitrile cosolvent (Figure 2) and found to be 100% at 48 hours in the presence of the cosolvent, which is not as prone to interference from compound precipitation during the experiment.

Figure 2

Effect of cosolvent on the apparent stability of ML283.

Synthetic Route

The probe compound SID 114279600 (CID 50930730) and numerous analogues were synthesized by the reaction sequence shown in Figure 3. Thus, the isolated, pure dimeric component from the dye primuline, P2 (CID 44251428) was diversified with various acid chlorides or other activated coupling partners.

Figure 3

General synthetic route for preparing probe compound SID 114279600 (CID 50930730) and associated analogues.

Submission of Probe and Five Related Analogues to the MLSMR

Samples of the probe compound and supporting analogues were shipped to the MLSMR on December 12, 2011.

Table 4Probe and analogue submissions to MLSMR (BioFocus DPI) for NS3 helicase inhibitor discovery

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Probe - ML283 (CID 50930730)
Probe/Analogue	Internal KU ID	MLS_ID (MLSMR)	CID	SID	Source (vendor or KU syn)	Amt (mg)	Date ordered/Submitted
Probe ML283	KUC108937A-02	MLS003882779	50930730	131341901	KU syn	21.8	12/12/11
Analogue 1	KUC107956A-02	MLS003882780	49849293	131341962	KU syn	21.2	12/12/11
Analogue 2	KUC107965A-03	MLS003882781	49849294	131341963	KU syn	23.3	12/12/11
Analogue 3	KUC108938A-02	MLS003882782	50930749	131341964	KU syn	21.4	12/12/11
Analogue 4	KUC108934A-02	MLS003882783	50930737	131341965	KU syn	23.0	12/12/11
Analogue 5	KUC110001N-02	MLS003882784	53356656	131341966	KU syn	23.3	12/12/11

Figure 4Structures of the probe candidate and supporting analogues

2.3. Probe Preparation

General synthesis and analysis experimental details: All reagents were used as received from commercial suppliers. The ¹H and ¹³C spectra were recorded on a Bruker Avance 400 MHz or 500 MHz spectrometer. Chemical shifts are reported in parts per million and were referenced to residual proton solvent signals. Flash column chromatography separations were performed using the Teledyne Isco CombiFlash R_F using RediSep R_F silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum inorganic hard layer with fluorescence). TLC plates were developed using iodine vapor. HPLC/MS analysis was carried out with gradient elution (5% CH₃CN to 100% CH₃CN) on an Agilent 1200 RRLC with a photodiode array UV detector and an Agilent 6224 TOF mass spectrometer (also used to produce high resolution mass spectra). Purification was carried out by Mass Directed Fractionation with gradient elution (a narrow CH₃CN gradient was chosen based on the retention time of the target from LCMS analysis of the crude sample) on an Agilent 1200 instrument with photodiode array detector, an Agilent 6120 quadrupole mass spectrometer, and a HTPAL LEAP autosampler. Fractions were triggered using an MS and UV threshold determined by HPLC/MS analysis of the crude sample. The conditions for HPLC analysis included the following: Waters BEH C-18, 1.7 μm, 2.1 × 50mm column; 0.6 ml/min flow rate; and pH 9.8 NH₄OH aqueous mobile phase. The conditions for purification included: Waters XBridge C18 5μm, 19 × 150mm column; 20 ml/min flowrate pH 9.8 NH₄OH aqueous mobile phase.

The probe compound was synthesized using the following protocols:

2′-(4-(3-Chlorobenzamido)phenyl)-6-methyl-[2,6′-bibenzo[d]thiazole]-7-sulfonic acid: To a solution of P2 (CID 44251428) (9.9 mg, 0.022 mmol) in pyridine (1 mL) at 80 °C, was added 3-chlorobenzoyl chloride (5.8 mg, 0.033 mmol, 1.5 equiv.). The reaction mixture was stirred at 80°C overnight. The product was isolated via reverse phase preparative HPLC to give a yellow solid (6.6 mg, 0.011 mmol, 51% yield).

3.1. Assay Development and Screening

To facilitate HCV helicase inhibitor identification, Belon & Frick developed a new helicase assay that simultaneously identifies compounds that interact with the helicase substrate and compounds that inhibit helicase action (14). This molecular beacon-based helicase assay (MBHA) uses a dual-labeled hairpin-forming DNA oligonucleotide annealed to a longer oligonucleotide, which forms a tail for the helicase to load (Figure 5). Once ATP is added, the helicase displaces the molecular beacon, resulting in a decrease in substrate fluorescence. By comparing substrate fluorescence before ATP is added (F₀), one can identify compounds that bind the MBHA substrate. At the same time, compounds that inhibit helicase action can be identified by fluorescence changes in an MBHA before and 30 (or 60) minutes after ATP addition (F₃₀/F₀ or F₆₀/F₀ ratio). In other words, the MBHA can be used as an “internal” counterscreen to identify compounds that appear to affect unwinding because they interfere with the assay. The most common types of interfering compounds are those that fluoresce or absorb light at the same wavelengths as the MBHA Cy5-labeled substrate. Alternatively, other compounds may bind the DNA substrate and distort it to change how the quenching moiety on the beacon is oriented relative to the Cy5 fluorophore.

Figure 5

Schematic drawing of the MBHA mechanism.

To identify HCV helicase inhibitors, the MBHA was used to screen the National Cancer Institute Developmental Therapeutics Program’s Mechanistic Set Library (http://dtp.nci.nih.gov/branches/dscb/mechanistic_explanation.html). In total, 827 compounds (at 20 μM) were screened using a MBHA with a DNA substrate (Figure 6 and Appendix D). When compound interference is plotted versus percent inhibition (Figure 7), it is clear that the majority of compounds that appeared to inhibit HCV helicase also quenched fluorescence of the MBHA substrate. Compound interference in the MBHA was evaluated by comparing the fluorescence of assays containing each compound to the fluorescence of DMSO-only negative controls before the addition of ATP. Hits were therefore defined as those compounds that did not interfere with the assays more than 20%, of which twelve were identified. These twelve hits were then subjected to a counterscreen that was designed to independently identify compounds that exhibit DNA-binding properties using a modified fluorescent intercalator displacement (FID) assay (22). The FID counterscreen used ethidium bromide to determine a molecule’s ability to bind DNA, and is based on the assumption that a DNA binding compound would displace a fluorescent DNA intercalating agent, leading to an observable decrease in observed fluorescence. Compounds were tested at 1.5 μM in the presence of the 25 base pair substrate used in the helicase assays (Figure 7). Results showed that even at a compound concentration 13-times lower than that used in the MBHA, most of the hit compounds decreased the fluorescence of an ethidium bromide-DNA complex by more than 10%, indicative of the molecule’s ability to bind DNA. The DNA minor groove-binding compound Berenil (EC₅₀=1.6±0.1 μM) was used a positive control in all FID assays (23).

Figure 6

Compound attrition of the NCI Mechanistic Set library during screening phases.

Figure 7

(A) The NCI Mechanistic Set of 827 compounds was screened with an MBHA, each at 20 μM. Fluorescence was read before and 30 minutes after ATP addition and compound inhibition was calculated. Hits were defined as compounds inhibiting more than 50% (more...)

Four compounds decreased the fluorescence of DNA-bound ethidium bromide less than 8%. The first, CdCl₂, was a known HCV helicase inhibitor that binds in place of the magnesium ion needed for ATP hydrolysis to fuel unwinding (24). The second, ellipticine, was found to fully quench DNA-bound ethidium bromide fluorescence at higher concentrations, and the IC₅₀ value for ellipticine in MBHAs (5.6±0.8 μM) was similar to its apparent affinity for DNA, suggesting that it inhibited the helicase by interacting with the substrate. Chromomycin A3 inhibited HCV helicase catalyzed-DNA unwinding with an IC₅₀ of 0.15 ± 0.03 μM, but had no effect on HCV helicase catalyzed RNA unwinding (data not shown). This false positive can be explained by the fact that Chromomycin A3 functionally resembles ethidium bromide as they both represent fluorescent DNA binding compounds (25). This result also demonstrates that not all DNA binding compounds will decrease DNA-bound ethidium bromide fluorescence in an ethidium bromide-based FID assay. The final sample, thioflavine S (CID 415676), did not affect DNA-bound ethidium bromide fluorescence until its concentration exceeded 100 μM, at which point a 20% fluorescence decrease was observed (Figure 11A). In dose response experiments, thioflavine S (CID 415676) inhibited HCV-helicase catalyzed DNA unwinding with an IC₅₀ of 16±4 μM, and it inhibited HCV catalyzed RNA unwinding with an IC₅₀ of 12±2 μM.

Figure 11

Compound interaction with the partially duplex helicase DNA substrate. (A) The ability of thioflavine S to quench the fluorescence of a ethidium bromide-stained DNA substrate. Berenil and another known DNA binding compound, ellipiticine, are included (more...)

Thioflavine S (CID 415676) is not a single compound but rather a heterogeneous dye that is structurally related to another heterogeneous yellow dye, primuline (CID 415713) (24, 26). Primuline (CID 415713) inhibited HCV helicase in MBHAs with similar potency (11±1 μM) as thioflavine S (CID 415676). To better understand how these dyes inhibit HCV helicase, both mixtures were separated using reverse-phase preparative HPLC or a combination of normal phase silica gel column chromatography and reverse-phase preparative HPLC.

The structure of commercial samples of thioflavine S (CID 415676) is not reported consistently, or is left intentionally vague, creating confusion over the chemical identity of the screening hit. For instance, the MSDS (Sigma Aldrich) for thioflavine S describes the compound only as “methylated, sulfonated primuline base.” In the NCI and PubChem online databases, thioflavine S (CID 415676, SID 550242, NSC71948) was reported as a mixture of methylated benzothiazoles, and Packham’s group also reported the same structure (27). To identify the components of commercially obtained thioflavine S (CID 415676) responsible for the observed activity, we purified the commercial sample (Sigma cat. #T1892) via reverse-phase preparative HPLC chromatography to obtain two compounds (T1 (CID 44251429) and T2 (CID 44251431)), the structures of which were ascertained using NMR, and LC/MS (Figure 8).

Figure 8

Structures of the isolated components of thioflavine S (T) and primuline (P).

Contrary to our expectation that the isolated compounds would be methylated primuline derivatives, isolated T1 (CID 44251429) and T2 (CID 44251431) were the N, N-diethyl products of the primuline monomeric and dimeric benzothiazoles. Of these, only T2 (CID 44251431) manifested inhibitory activity against helicase-catalyzed DNA unwinding (Table 5). Encouraged by these results, the dye primuline (CID 415713, MP Biomedicals cat. #195454) was also purchased and purified. Primuline (CID 415713) inhibited HCV helicase catalyzed DNA unwinding with about half the potency as T2 (CID 44251431) (Table 5).

Table 5

Isolated thioflavine S and primuline components for NS3 helicase inhibitor discovery.

In total, six compounds were isolated from primuline (CID 415713). The two major components, P1 (CID 44251427) and P2 (CID 44251428), were separated via reverse-phase preparative HPLC in 9.2% and 7.6% isolated yield (by weight) for P1 (CID 44251427) and P2 (CID 44251428), respectively. In the MBHA, P2 (CID 44251428) was equipotent to the primuline mixture (CID 415713), while P1 (CID 44251427) was inactive. That the purified major active component P2 (CID 44251428) did not possess increased potency compared to the mixture containing the inactive P1 (CID 44251427) was unexpected and hinted that highly potent components could be present in the primuline mixture in small amounts. The direct isolation of minor components via reverse-phase preparative HPLC of the dye mixture was not successful. Hence, the purification procedure was modified, enabling the isolation of four minor components. Four chromatographic bands enriched with minor components (UV and LC-MS) were obtained from silica gel chromatography of commercial primuline (CID 415713) upon elution with 20% DCM/MeOH. Subsequent reverse-phase preparative HPLC purification afforded the relatively minor components P1a (CID 44251433), P2a (CID 44251434), P3 (CID 44251436) and P4 (CID 44251437), where P3 (CID 44251436) and P4 (CID 44251437) represented 0.49% and 0.23% isolated yield (by weight) of the dye, respectively. All purified compounds were composed of a central benzothiazole oligomer of 1–4 units terminating with a p-aminobenzene group (Figure 8).

In the MBHA, all of the compounds purified from primuline were helicase inhibitors although P1 (CID 44251427) and P1a (CID 44251433), only partially inhibited unwinding at the highest concentrations tested (Figure 9B). Potency correlated directly with the length of the benzothiazole chain. For P3 (CID 44251436) or P4 (CID 44251437), only about 1 μM of either was needed to reduce the rate of helicase catalyzed DNA unwinding by 50% (Figure 9, Table 5).

Figure 9

Effects primuline and its components on HCV helicase catalyzed DNA unwinding. (A) MBHAs performed at various concentrations of primuline (CID 415713). Reactions were initiated by adding ATP at the indicated time. (B) Initial rates of DNA unwinding in (more...)

These purified compounds resemble molecules known to bind DNA, typically along the minor groove, such as the cyanine dye known as BEBO (CID 11741853) (Figure 10),(28, 29) but unlike many DNA binding benzothiazoles, these helicase inhibitors are not positively charged. Instead they are anionic, due to the sulfonate groups on the terminal benzothiazole rings.

Figure 10

Structures of representative DNA binding agents.

To test if the primuline and thioflavine S components interact with DNA, FID assays were repeated with each. Again, ethidium-bromide-stained MBHA-substrate (i.e. the same sequence as that shown in Figure 5, but without the Cy5 and IBQ modifications) florescence was read before and after compound addition. Far more thioflavine S was needed in this assay to influence ethidium-bromide-stained DNA fluorescence than known DNA-binding compounds (Figure 11A). In contrast to the parent dyes, less of the dye components were needed to effect ethidium-bromide-stained DNA fluorescence. All compounds, except T1 (CID 44251429), P1 (CID 44251427) and P1a (CID 44251433), decreased the fluorescence of ethidium-bromide-bound DNA by at least 10% when present at 100 μM. However, only compounds P3 (CID 44251436) and P4 (CID 44251437) decreased ethidium-bromide–bound DNA fluorescence more than 50% at the highest concentration tested (Figure 11B).

Because P3 (CID 44251436) and P4 (CID 44251437) clearly interacted with ethidium-bromide-stained DNA, we suspected that the other dye components might also bind DNA, but in ways that do not displace the intercalated ethidium bromide. We therefore examined the effect of each compound on DNA stained with other dyes, and found that most compounds decreased the fluorescence of DNA stained with SYBR Green I (CID 10436340). The affinity of primuline (CID 415713), thioflavine S (CID 415676) and the purified compounds for the MBHA substrate DNA was therefore estimated using a modified FID assay where ethidium bromide (CID 14710) was replaced with SYBR Green I (CID 10436340). Less of each compound was needed to decrease the fluorescence of SYBR Green I stained DNA, and none of the compounds appeared to bind DNA more tightly than the potency with which they inhibited the helicase reaction. As seen in other assays P4 (CID 44251437) bound more tightly than all other compounds (Figure 11C, Table 5).

In addition to inhibiting the helicase reaction and binding the helicase substrate, the dye components possess unique properties that make them uniquely useful molecular probes. First they are fluorescent, and their fluorescence can be used to monitor the compounds both in vitro and in vivo as is done both with thioflavine S (30) and primuline (31). Second, they inhibit other NS3 activities, namely the protein’s ability to cleave ATP, the fuel for the helicase reaction, and the peptides via the protease domain. Though numerous NS3 protease inhibitors have been developed, none have yet been reported which inhibit both the NS3 protease and the NS3 helicase functions.

The compounds isolated from the two yellow dyes are all fluorescent, absorbing light around 350 nm, and emitting near 420 nm (Figure 12). Their extinction coefficient and peak absorption wavelengths increase as the length of the benzothiazole oligomer increases. Their relative fluorescence decreases with the length of the benzothiazole chain. None of the compounds absorbed light near the absorption or emission wavelengths of the Cy5-labeled MBHA substrate, or the wavelengths where ethidium-bromide-stained DNA, SYBR Green I (CID 10436340) stained DNA, NS3-catalyzed peptide cleavage, or ATP hydrolysis were measured.

Figure 12

Absorbance and fluorescence emission spectra of primuline, thioflavine S, and their individual components.

Compounds can inhibit HCV helicase-catalyzed ATP hydrolysis by competing with either ATP or the nucleic acid that stimulates ATP hydrolysis. To test if thioflavine S inhibits the ATPase and examine where thioflavine S might act, initial rates of ATP hydrolysis were measured in the presence of various concentrations of ATP, RNA and thioflavine S. Thioflavine S did not compete with ATP (data not shown) but partially compete with RNA (Figure 13A). More RNA was needed to stimulate ATP hydrolysis as thioflavine S concentration increased. However, unlike compounds that bind in place of RNA (or DNA), thioflavine S retained inhibitory potential even when the protein was saturated with RNA (32). In addition, thioflavine S inhibited HCV helicase catalyzed ATP hydrolysis even in the absence of RNA, suggesting a direct interaction between the protein and thioflavine S.

Figure 13

Effects of thioflavine S and the most potent primuline component on HCV helicase catalyzed ATP hydrolysis. (A) ATPase assays were performed in presence of indicated concentrations of thioflavine S and poly(U) RNA (measured as μM UMP). ATP was (more...)

The most potent primuline component, P4 (CID 44251437) was then added to NS3 ATPase assays with and without RNA. P4 (CID 44251437) inhibited ATP hydrolysis both in the presence and absence of RNA (Figure 13B), indicating that the compound is not simply sequestering RNA and preventing activation of ATP hydrolysis. It is also important to note that far more P4 (CID 44251437) is needed to inhibit ATP hydrolysis in the absence of RNA, indicating that the presence of nucleic acid enhances the enzyme’s affinity for the inhibitor (Figure 13C). When ATPase assays were performed with compounds isolated from thioflavine S and primuline (Table 5) the same pattern was observed as previously seen in the MBHAs and FIDs (i.e. the longer benzothiazole oligomers were always more potent in all assays than the shorter oligomers).

It is not uncommon for helicase inhibitors to inhibit the protein’s ability to hydrolyze ATP since ATP hydrolysis is needed to fuel unwinding. However, few compounds have been reported that inhibit the helicase and the protease function of NS3. Surprisingly, P4 (CID 44251437) and some of the other compounds purified from thioflavine S and primuline also inhibited NS3 catalyzed peptide cleavage (Figure 14). The compounds inhibited the protease if they were added before (Figure 14A) or after the protease substrate. The later experiment demonstrates that inhibition is not simply due to the compound quenching the fluorescence of the protease reaction product. The potency with which the primuline and thioflavine components inhibited the NS3 protease mirrored the potency seen in helicase assays (Table 5).

Figure 14

Effects of primuline and P4 (CID 44251437) on NS3 catalyzed peptide cleavage. Assays were performed at 23 °C using the SensoLyte® 520 HCV Protease Assay Kit (Anaspec) with 20 nM of a purified recombinant single chain NS3/NS4A protein (33). (more...)

3.2. Dose Response Curves for Probe

Figure 15Dose response curves for probe and two analogues

Effect of CID 50930730/ML283 on standard MBHAs (A) compared with a slightly more active compound CID 49849293 (B) and less active compound CID 50930749 (C). MBHAs were performed in the presence of 16 concentrations of each compound ranging from 100 μM (black), 67 μM (blue), 45 μM (red), 30 μM (green), 20 μM (magenta), 14 μM (light blue), 9 μM (orange), 6 μM (purple), 4 μM (grey), 2.7 μM (pale blue), 1.8 μM (peach), 1.2 μM (dark green), 0.8 μM (pink), 0.5 μM (light purple), 0.4 μM (light orange), and 0.25 μM (pale magenta). Reactions were initiated after 60 seconds by adding ATP to a final concentration of 1 mM. Data obtained after ATP addition are fit to a first order decay equation to calculate initial rates in the presence of various concentrations of each compound. (D) Initial unwinding rates plotted versus compound concentration. Velocities are fit to a dose response equation as described in Appendix A.

3.3. Scaffold/Moiety Chemical Liabilities

The probe compounds based on an oligomeric benzothiazole scaffold appeared to be relatively stable based on our observations from day-to-day handling related to their synthesis, analysis, dissolution-transfer, lyophilization, and storage. The probe compounds do not contain moieties that are known or expected to be generally reactive.

3.4. SAR Tables

The positive results obtained with the higher-order components P3 (CID 44251436) and P4 (CID 44251437) isolated from primuline (CID 415713) led us to undertake a structure–activity relationship study based on them. However, difficulties with both isolation of sufficient quantities from the commercial sources and with fully synthetic approaches necessitated that we consider more accessible bioisosteric equivalents. Accordingly, we focused on modification of the amino group at the terminus of P2 (CID 44251428), which was available in sufficient quantities to serve as a starting material. We envisioned that an appropriately rigid linker could substitute for the third benzothiazole ring of P3. Thus, the terminal amine of P2 (CID 44251428) was acylated to give amides, reacted with isocyanates or sulfonyl chlorides to give urea analogues and sulfonamides, respectively (Scheme 1 and Figure 16). Table 6 illustrates the effect of various functional groups on substituted phenyl amide derivatives, the most extensively investigated modification strategy. These included electron-donating or -withdrawing groups as well as aliphatic substitutions. The unsubstituted benzamide analogue (CID 49849280, entry 11) possessed modest potency in assays employing helicase enzyme from both the 1b(con1) and 2a(JFH1) HCV genotypes (IC₅₀ = 10.7 ± 1.5 μM and 4.6 ± 1.4 μM, respectively). Introduction of a diverse range of para substitution (e.g. NH₂ (CID 49849300), N(CH₃)₂ (CID 49849284), OCH₃ (CID 49849282), CO₂CH₃ (CID 49849290), t-Bu (CID 49849299) and Br(CID 50930740)) had no significant effect on the potency of helicase inhibition. Improved analogues were obtained when Cl (CID 49849302), F (CID 49849294), CH₃ (CID 49849286) or CF₃ (CID 49849276) were tested in the para position. The increased size of alkyl substitution from Me to t-Bu resulted in an ca. 2-fold activity loss. Altering the Cl position from para to meta (entries 19 and 22, CID 49849302 and CID 50930730) had no effect on helicase inhibition, although the meta substitution displayed weaker DNA binding coupled with promising cell-based HCV replicon values. Increasing, the number of chloro groups as in CID 50930737 (entry 23) showed no improvement. The CF₃ substituted series possessed an intriguing range of activity. The 4-CF₃ (CID 49849276) analogue was the most potent compound in the helicase assays (IC₅₀ = 1.8 ± 0.4 μM and 2.5 ± 1.0 μM, for 1b(con1) and 2a(JFH1) HCV genotypes respectively). Moving the CF₃ group from the para position to the ortho or meta position resulted in a loss of inhibitory activity (7–10 fold, entries 24–26, CID 50930748, CID 50930755 and CID 49849276). Introduction of additional fluorine or CF₃ substitution on the derivatives also exhibited no effect on the helicase inhibition when compared to the 4-CF₃ (CID 49849276) substituted compound (see entries 27–33, CID 50930749, CID 50930745, CID 50930751, CID 50930741, CID 50930743, CID 50930732 and CID 50930733). In fact, depending on the position of fluorine or CF₃ substitution, a significant decrease in potency was observed. Alternate amide linked moieties to the substituted benzamides were briefly explored (entries 34–44, Table 7). Replacing substituted phenyl with a methyl alkyl group resulted in complete loss of activity (CID 53239937, entry 44). Analogues targeting improved solubility by replacing the phenyl ring with pyridine ring, produced less potent analogues (2–5 fold decrease in helicase activity, CID 50930756 and CID 50930734, entries 40 and 41). Though significantly less potent, these analogues did possess greatly improved solubility under the assay conditions; for example CID 50930756 was soluble at > 100 μM compared to ML283, which possessed a solubility of 29.2 μM (Table 10). N-methylation of the naphthyl analogue (CID 49849293) also caused a significant drop in activity (5 fold,

Scheme 1

Reagents: (a) substituted benzoyl chloride, pyridine, 80 °C; (b) arylisocyanate, DMF, 80 °C; (c) arylsulfonyl chloride, pyridine, 80 °C.

Figure 16

Structures of derivatives based on the major components in primuline.

Table 6

SAR analysis of primuline P2 amide derivatives for NS3 helicase inhibitor discovery.

Table 7

SAR analysis of additional P2 derivatives for NS3 helicase inhibitor discovery.

Table 10

Comparison of solubility and potency for ML283 and selected analogues.

CID 52914816), which could indicate the loss of a key hydrogen bond interaction. While none of these analogues possessed improved helicase activity compared to ML283, the solubility of the 3-pyridyl analogue (CID 50930756) and nascent potency of the naphthyl analogues (CID 49849293 and CID 53308659) provide inspiration for designing future analogue sets.

In an effort to mimic the tetrameric structure of P4 (CID 44251437), the more elaborate amide derivatives CID 49849289 and CID 49849287 (entries 38 and 39, Table 7) were synthesized. No improvements in potency were observed for these tetrameric analogues over the previous trimeric analogues. The simple one-step synthesis of the trimeric analogues compared to the tetrameric analogues prompted us to focus on the former for future studies targeting more potent inhibitors of helicase function and HCV replication.

In DNA binding assays, none of the P2 derivatives decreased the fluorescence of DNA-bound ethidium bromide by more than 10% even at 100 μM. However, like P3 (CID 44251436) and P4 (CID 44251437), many of the derivatives appeared to bind SYBR-Green-I-stained DNA, and these EC₅₀ values in SYBR Green-based FID assays are reported. Most of the amide derivatives were at least 10-fold more potent in the MBHA than the DNA-binding assay. The 3-Cl analogue (ML283, CID 50930730) was chosen for nomination as the probe candidate based on a combination of helicase assay inhibition (both 1b(con1) and 2a(JFH1) HCV genotypes), high percentage of cellular HCV replication (54% at 10 μM) coupled with no detectable cytotoxicity and low DNA binding in the SYBR Green FID assay (EC₅₀ > 100 μM).

Two additional classes of chemical linkers were also explored to mimic the benzothiazole moiety (Table 7). The urea analogues, which were synthesized from P2 (CID 44251428) by reacting it with different isocyanates and the sulfonamide analogues prepared via the sulfonation of P2 (CID 44251428) with sulfonyl chlorides. The p-tolyl urea analogue CID 49849298 (entry 46) has potency in the MBHA and replicon assays approaching that of the probe candidate, although increased DNA binding was observed (17 μM for CID 49849298 compared to >100 μM for ML283). Slightly less potent analogues were achieved via sulfonation (e.g. CID 49849295, 2-fold activity drop) compared to ML283.

The other major component isolated from the primuline mixture, P1 (CID 44251427) was also investigated as a substrate for the synthesis of active analogues. Table 8 summarizes the derivatives synthesized based on the monomer P1 (CID 44251427). None of the compounds showed significant activity in the replicon assay and only the Fmoc-protected amines CID 53377547 and CID 53377543 (entries 56 and 60) and the trifluormethylphenyl analogue CID 53308658 (entry 58) possessed MBHA potency approaching that of ML283. The absence of replicon activity for this series was the primary motivation to pursue derivatives based on the equally abundant P2 (CID 44251428).

Table 8

SAR analysis of P1 derivatives for NS3 helicase inhibitor discovery.

The synthetic diversification strategy of the isolated component P2 (CID 44251428) allowed us to produce analogues with a diverse range of substitution on the ‘eastern’ portion of the compounds in quantities > 20 mg in one or two synthetic steps. A strategy capable of incorporating modifications anywhere in the molecule and which did not depend on the separation of a complex dye mixture to obtain starting material would be highly advantageous, if the synthetic manipulations could be minimized. All attempts toward the de novo synthesis of oligomeric analogues of P3 (CID 44251436) and P4 (CID 44251437) were unsuccessful despite a dedicated effort. Inspired by the success of the amide derivative of P2 (CID 44251428) in the MBHA and replicon assay, a series of simplified analogues were synthesized. Table 9 summarizes the structures and activities of these compounds. Though active in the replicon assay, they were also found to be marginally cytotoxic. The promising results in the semisynthetic series and increasing complexity of the synthetic route to the target analogues prompted us to focus on the semisynthetic series.

Table 9

SAR analysis of selected fully synthetic analogues for NS3 helicase inhibitor discovery.

The SAR campaign to identify ML283 was based on a total of 55 compounds in addition to the eight isolated dye components. Figure 17 illustrates the different structural elements explored in the course of the search for an accessible analogue of P3 (CID 44251436), and which positions tolerated modification. While the majority of analogues were based on the derivatization of the primary amine on P2 (CID 44251428), this was a highly fruitful site for diversity, affording analogues across the potency range of 1.8 to > 100 μM in the NS3 helicase assay. Furthermore, all compounds were well tolerated in the cell viability assay yet varied in their ability to inhibit the HCV replicon within the range of 0.3 to 64%. All 55 SAR compounds are novel chemical entities and we have filed a provisional patent application for these compounds and their application as inhibitors of helicase function and HCV replication. In fact, a SciFinder substructure search for any carbonyl derivatives of P2 (CID 44251428) did not return any hits (Figure 18), indicating the novelty of this derivatization approach. To more fully assess the novelty of the probe and analogues as inhibitors of the NS3 helicase, we conducted a SciFinder substructure search on the dimeric primuline component P2 (CID 44251428), which was the starting material for the majority of the analogues. The search results indicated that 116 substances or mixtures contained this substructure, as reported in 88 patents and references. Of these references and patents, 63 focused on the synthesis or use of the substances as inks or dyes for textiles, outside of a biological context. The 25 remaining references were largely focused on utilizing these substances or dye mixtures for the identification of organisms and/or molecular structures and no instances of using primuline components as helicase enzyme inhibitors or for the treatment of HCV were found. The full search results are discussed in Appendix E.

Figure 17

Summary of the SAR strategy to synthesize analogues based on the hit, P3.

Figure 18

P2 and amide derivative substructures used in SciFinder searches.

3.5. Cellular Activity

Two cell-based assays have been completed to characterize CID 50930730/ML283 and most analogues, as reported in the SAR tables. These assays were: 1) a Renilla luciferase reporter assay to examine the ability of compounds to inhibit replication of a HCV Renilla luciferase subgenomic replicon and 2) a Firefly luciferase-based viability assay that measures the effect of compounds on cell viability. Both assays were conducted on HCV replicon containing cells treated with compounds at 10 μM. The activity for the probe compound in both cell-based assays is described in detail in Section 3.4, SAR Tables, and detailed assay descriptions are given in Section 3.1.

The HCV replicon chosen (Figure 19A) was derived from the same HCV strain (con1, genotype 1b) as the NS3 protein used for screening and enzyme assays, and was a variant of the replicon first reported by Lohmann et al. (34) with two cell culture adaptive mutations (E1202G and S2204I) (35, 36). This subgenomic replicon also had a Renilla luciferase gene fused to the 5′-end of the neomycin phosphotransferase gene used for selection, so that the cellular levels of Renilla luciferase correlated directly with the amount of HCV RNA present in cells (37). After replicon transfection and selection, cells were treated in parallel in two triplicate sets. One set of cells was used for Renilla luciferase assays and the other set was used to determine cell viability using a firefly luciferase-based assay and all compounds were tested at 10 μM.

Figure 19

Effect of ML283, CID 50930749, CID 486270, and Telaprevir on Huh7.5 hepatoma cells harboring a stably transfected subgenomic rLuc HCV replicon. (A) Schematic genome map of the HCV replicon used in this project. Percent Renilla luciferase remaining (circles) (more...)

CID 50930730/ML283 and many of its close analogues showed some modest activity against the HCV replicon, but effects varied depending on the batch of cells used and the passage number of stably transfected replicon cells. We observed also that cell-based experiments with CID 50930730/ML283 are difficult due to its limited solubility in PBS and cell culture media (see below) and because of its ability to aggregate at higher concentration in the absence of non-ionic detergents (e.g. Tween20). This observation has limited our efforts to study the dose dependent efficacy of CID 50930730/ML283 because typically no more than 10 μM of CID 50930730/ML283 can be administrated before it begins to appear to aggregate in the culture (Figure 19A). Experiments with other, more soluble analogues yielded more reproducible concentration response curves. For example, CID 50930749 showed a concentration-dependent inhibition of replicon luciferase activity with 5 ± 2 μM needed to reduce replicon-encoded luciferase by 50%. Even at the highest concentration tested (50 μM) CID 50930749 was not toxic to Huh7.5 cells (Figure 19C). In the same system, the most potent comparison helicase inhibitor (the symmetrical benzimidazole CID 486270, had a IC₅₀ of 8 ± 4 μM but it decreased cell viability with a CC₅₀ of 87 ± 40 μM (Figure 19D). An approved HCV drug, Telaprevir, inhibited the replicon with an IC₅₀ of 0.62 ± 0.05 μM and reduced cell viability with a CC₅₀ value of 28 ± 5 μM (Figure 19E).

To further examine the anti-HCV activity of CID 50930730/ML283 in cells containing HCV replicons, we examined the effect of CID 50930730/ML283 and CID 50930749 exposure on HCV RNA levels. Assays with the probe CID 50930730/ML283 were again confounded by its poor solubility in cell culture media, as evidenced by the fact that concentration dependent reduction of HCV RNA was reversed after the concentration of CID 50930730/ML283 exceeded 12 μM. In contrast, CID 50930749 exposure reduced HCV RNA levels in replicon cells in a concentration-dependent manner (Figure 20A). To compare the effect of CID 50930749 to that of interferon α/2b, the current standard of HCV care, cells were exposed to each for ten days. Analysis of luciferase activity (not shown) and HCV RNA (Figure 20B) during, and at the end of treatment, showed that CID 50930749 reduced HCV content in a time-dependent fashion. In repeated experiments CID 50930749 led to a 16-fold decrease in HCV replicon RNA content. This was about one-quarter of the effect seen with interferon.

Figure 20

Effect of ML283, CID 50930749, and interferon on cellular HCV RNA levels. (A) Amount of HCV RNA remaining in cells exposed to indicated amounts of ML283 or CID 50930749. RNA was measured using 1 μg of total RNA with specific Taq-man probes and (more...)

More extensive cell based assays have been completed with CID 50930749, which again was the probe analogue that was most active in cells (In vitro, CID 50930749 was less potent and less specific than CID 50930730/ML283). For example, CID 50930749 was used to investigate how probe treatment might affect the cellular location of HCV replication complexes. In replicon containing Huh7.5 cells, HCV replication occurs in a membranous web associated with the rough endoplasmic reticulum (38). Clear differences in such complexes were observed when mock-treated (DMSO only) cells were compared with cells treated with CID 50930749, primuline, or interferon-α/2b. Like interferon, CID 50930749 treatment reduced the number of replication complexes (Figure 21). In both cases, only reticular staining of NS5A and no replication complexes were seen. However, mock-treated cells and primuline-treated cells showed no apparent differences in HCV replication complexes. These results support the anti-HCV activity of CID 50930749.

Figure 21

Effect of CID 50930749 on the cellular location of HCV Replication complexes seen in the replicon-containing Huh7.5/Con1sg-Rluc cells. After 72 hours of mock treatment with 0.5% DMSO (A), 100 units of interferon (B), 10 μM primuline (CID 415713) (more...)

Since CID 50930749 is fluorescent, it can be directly observed in cells as shown in Figure 22. HCV-replicon-bearing Huh7.5 cells were treated with CID 50930749 for 72 hours and cells were fixed, permeabilized, and stained for NS5A. Cells were imaged for CID 50930749 localization and compared with the antibody stained NS5A replication complexes. NS5A and CID 50930749 do not appear to co-localize, but since the viral target of CID 50930749 is HCV helicase, ongoing experiments aim to look at co-localization of CID 50930749 with viral helicase using specific anti-helicase mAb and higher resolution imaging. Interestingly, CID 50930749 showed a diffused and dotted cytoplasmic staining patterns in replicon cells and no evidence of CID 50930749 getting in the nucleus was observed.

Figure 22

CID 50930749 location in cells. Huh7.5/Con1sg-Rluc cells were treated with CID 50930749 for 72 hours, permeabilized and stained with 9E10 α-NS5A antibody and Alexa 546 secondary antibody. (A) Localization of CID 50930749 fluorescence observed (more...)

The data above show that cellular experiments with the probe compound, which is a more potent and specific helicase inhibitor, might be possible if administered with a compatible excipient. Once a system is found to deliver the probe to cells, the probe and less specific analogues such as CID 50930749 could serve as a powerful tool to yield insights into when and where the helicase acts in an infected cell, or to test the hypothesis that the helicase activity on RNA or DNA is needed for HCV replication. Furthermore, if our ongoing experiments at investigating direct interaction of HCV helicase and these fluorescent probes in cell are successful, the probe could then be used to directly monitor virus morphogenesis and egress in real time using live cell imaging. Preliminary work with CID 50930730/ML283 shows that, like CID 50930749, CID 50930730/ML283 enters cells and specifically stains certain cellular structures (Figure 23).

Figure 23

Use of fluorescence microscopy to visualize CID 50930730/ML283 in cells. (A) Cells exposed to ML283 show specific cell staining, and the compound appears to stains cytoplasmic structures, with faint or no staining of nucleus. No staining was observed (more...)

3.6. Profiling Assays

The probe ML283 and a series of analogues were screened for solubility under conditions similar to those used in the MBHAs (Table 10, MOPS buffer). Analogues targeting improved solubility by replacing the phenyl ring with pyridine ring, produced less potent analogues (2–5 fold decrease in helicase activity, CID 50930756 and CID 50930734, entries 40 and 41, Table 10). Though significantly less potent, these analogues did possess greatly improved solubility under the assay conditions; for example CID 50930756 was soluble at > 100 μM compared to ML283, which possessed a solubility of 29.2 μM (Table 10). Future SAR efforts will focus on combining the solubility enhancing structural elements in analogues which retain potency for the NS3 helicase. To exclude the possibility that compound aggregation was the source for the observed helicase inhibition, the solubility of the probe candidate and several analogues was measured in a mock assay medium containing BSA, detergent (Tween 20) and DMSO (Table 10, assay matrix). These data demonstrate that the probe is sufficiently soluble (29.2 μM) to fully account for the observed helicase inhibition.

Another physical property of the probe and all its analogues that has been examined is their optical properties. Compared to P4 (CID 44251437), CID 50930730/ML283 has a sharper absorbance peak centered at 360 nm (Figure 24A). When excited at 350±12 nm, CID 50930730/ML283 is also more fluorescent than P4 (CID 44251437).

Figure 24

Spectra of CID 50930730/ML283 and some analogues. (A) Absorbance of 20 μM aliquots of the indicated compounds. (B) Fluorescence emission spectra of compounds when excited at 350 ± 12 nm.

To uncover other off-target effects, the probe has been analyzed for its ability to influence the interaction of an unrelated nucleic-acid-binding-protein with DNA. The protein chosen was the single-stranded DNA binding protein (SSB) encoded by E. coli. SSB has been widely studied because of its ability to coordinate DNA replication and recombination and is commercially available (New England Biolabs). We designed a fluorescence polarization-based assay to monitor the binding of SSB to single-stranded DNA (Figure 25A). In the SSB assay, the polarization of a Cy5-labeled oligonucleotide is monitored. Oligonucleotide polarization increases when SSB is added to the assay. SSB increased the fluorescence polarization of Cy5-dT15, with about 20 nM SSB needed to saturate the oligonucleotide with a K_0.5 of 9.6 ± 2 nM in the presence of 5 nM Cy5-dT15. DNA-binding compounds like P4 (CID 44251437) cause SSB to fall from the DNA. The probe and its analogues were added to this assay each in a 16 point 1.5-fold dilution series starting at 100 μM, in order to calculate an IC₅₀ value for each. Clear differences are apparent with CID 50930730/ML283 displacing far less SSB than all other compounds tested (Figure 25B). Concentration response curves reveal that about 67-times more CID 50930730/ML283 (IC₅₀=200 μM) is needed to displace the same amount of SSB from DNA than is needed to inhibit HCV helicase in an MBHA (Figure 25C). These assays further support the notion that CID 50930730/ML283 is more specific than P4 (CID 44251437), or any other analogue, with regard to its ability to inhibit NS3 helicase. All data using this counterscreen are reported in the SAR tables.

Figure 25

Effect of compounds on the polarization of a Cy5-dT15-E. coli single-stranded DNA binding protein (SSB) complex. (A) FP based assay to monitor SSB binding to Cy5-dT15. (B) Ability of various primuline derivatives to inhibit HCV helicase (x-axis) and decrease (more...)

We have also analyzed the ability of most of the compounds in Table 10 to inhibit NS3 catalyzed ATP hydrolysis and peptide cleavage. IC₅₀ values for each assay are reported in Table 5 and Table 10. Importantly, the probe (CID 50930730/ML283) maintains an ability to inhibit the ATP hydrolysis that fuels unwinding, but it is a 50-fold less potent protease inhibitor than P4 (CID 44251437).

The pharmacokinetic (PK) properties of the probe (CID 50930730/ML283) were profiled using a standard panel of assays (Table 11). The most striking result is the solubility variation depending on the buffer system used. While the aqueous solubility is low in the PBS--based solvent system, in both the detergent-containing (Tween 20) assay matrix and the proprietary Prisma HT buffer system the compound is readily soluble. The unknown identity of the components in the Prisma HT buffer system complicates further speculation into the solubility discrepancy. The solubility results have inspired us to pursue improving compound solubility through formulation with other solubilizing agents and these experiments are currently in progress. The low solubility and permeability of the probe (CID 50930730/ML283) were not surprising for a polycyclic aromatic compound of molecular weight 592 g/mol. Encouragingly, the probe (CID 50930730/ML283) was highly stable under the various conditions screened and possessed no detectable hepatic toxicity. Analogues possessing improved solubility and PAMPA properties will be the aim of future efforts.

Table 11

Summary of in vitro ADME properties of ML283 CID 50930730.

4.1. Comparison to existing art and how the new probe is an improvement

One of the challenges in developing chemical probes that target helicases is that potent helicase inhibitors often exert their actions by binding nucleic acid helicase substrates. Such compounds lack specificity because they can inhibit any protein that needs to access the genetic material. We attempted to discover more specific helicase inhibitors that do not target nucleic acids using high throughput helicase and DNA-binding assays, but even the most promising compounds, which were purified from the most active library sample, interacted with the DNA substrate in the absence of protein. We have, nevertheless, been able to engineer potent analogues that interact with DNA less tightly yet still retain an ability to inhibit helicase catalyzed nucleic acid strand rearrangements. Some of these compounds retain the important ability to inhibit HCV replication in cells, and could therefore prove useful for antiviral drug development.

To discover helicase inhibitors that do not bind nucleic acids, we screened a compound library using a helicase assay that simultaneously detects a compound’s ability to interact with the helicase substrate and its ability to interfere with the movement of HCV helicase through DNA. The results of the screen confirmed that most library samples that block helicase movements also interact with the helicase substrate. We confirmed that most of the newly uncovered HCV helicase inhibitors interact with DNA using a DNA binding assay (Figure 7). The binding assay was based on a FID assay (22) that monitors fluorescence changes that occur when a compound interacts with DNA stained with ethidium bromide. The most active sample in the screened library that least affected the fluorescence of ethidium bromide-stained DNA was the yellow dye thioflavine S (CID 415676), and potent benzothiazole oligomers were purified from this dye and its relative, primuline (CID 415713). When the most active benzothiazole oligomers purified from primuline (CID 415713) were found to interact with SYBR Green I stained DNA, we learned that the ethidium bromide-based FID failed to detect the interaction of thioflavine S (CID 415713) with DNA. A more sensitive SYBR Green I assay was therefore developed and used to chemically optimize the probe by aiding the design of the more specific P2 derivatives. The observation that DNA-interactions escaped detection in the ethidium bromide-based FID reinforces the notion that care needs to be taken when using FID assays because DNA-binding compounds might not displace the bound intercalator. There is likewise still some uncertainty as to whether or not the derivatives that failed to influence the florescence of SYBR Green-stained DNA interact with nucleic acids. Preliminary results using isothermal titration calorimetry support the relative affinities reported here, but more extensive DNA- and RNA-binding experiments clearly need to be done with these compounds.

Figure 26Comparative effects of benzothiazoles in HCV helicase assays

Potency of each compound tested in the study in MBHAs is plotted against percent ethidium bromide DNA fluorescence quenched (FID) at 100 μM. Filled circles mark CID 50930730/ML283 and P4 (CID 44251437). Open Circles mark published helicase inhibitors used for comparison.

The most potent benzothiazoles were notably more effective than the recently reported helicase inhibitors used for comparison, two of which appeared to function primarily by interacting with the DNA substrate (CID 3084034 and CID 46913723). CID 50930730/ML283 was nearly as potent as P4, and it eliminated the HCV replicon without apparent toxicity similar to both P3 (CID 44251436) and P4 (CID 44251437). Also like P4 (CID 44251437), CID 50930730/ML283 inhibited HCV helicase catalyzed RNA unwinding and ATP hydrolysis (data not shown).

The compounds reported here are more potent and specific than other recently reported HCV helicase inhibitors (Table 12, Figure 27). Only CID 3084034 and CID 486270 inhibited MBHAs with potency similar to ML283, although the precise effects of CID 3084034 and CID 46913723 on HCV helicase action were difficult to assess because both interfered with the MBHA. In cells, some of comparison compounds inhibited the replicon like the probe and its analogues. Only CID 46202556 was more effective than the probe analogue that was most active in cells (CID 50930749). None of the comparison helicase inhibitors were particularly toxic at 10 μM except for CID 50904396.

Table 12

Activity of reference inhibitors under comparison assays.

Figure 27

Dose response curves for helicase inhibitors used for comparison. MBHAs were performed in the presence of the indicated concentrations of each compound. Reactions were initiated after 210 seconds by adding ATP to a final concentration of 1 mM. Data obtained (more...)

We have also not been able to reproduce all of the results previously reported for the comparison helicase inhibitors. For example, the nucleotide mimic CID 50904396, which had been reported to have a K_i of 20 nM (18) had almost no detectable effect on the MBHA at 5,000 times higher concentration (Table 12). While it had some antiviral activity, the activity was coupled with notable toxicity at 10 μM (Table 12). The acridone CID 46202556 was about 17-times less potent in our helicase assay than it was in a previously reported assay, but yet it still was one of the most effective compounds in eliminating the HCV replicon. Similarly, although its interaction with the helicase substrate obscured effects in our MBHA, the triphenylmethane CID 3084034 displayed no inhibition at its previously reported IC₅₀ of 12 μM, although the compound did not appear as cytotoxic as had been reported (20). CID 46913723 had antiviral activity as previously reported, but again, compound interference in the MBHA made its effect on the helicase in vitro difficult to judge (21).

To test if our different results were due to different assay conditions, we have repeated helicase assays with the above inhibitor using a helicase assay that did not rely on molecular beacons. Using the same assay that was designed by Boguszewska-Chachulska et al. (39) we found that all four comparison compounds were about 5 times more potent, but they were still 5–10 times less potent than the probe or its most potent analogues. Interestingly, we found that the activity for the acridone (CID 46202556) was pH dependent and it only inhibited unwinding above pH 7. Also the nucleotide mimic (CID 50904396) only appears to inhibit the reaction when MgCl₂ was substituted with MnCl₂. The effect of all four comparative compounds is enhanced when reaction temperatures are increased from 23 °C to 37 °C where the DNA is less stable and more easily unwound by the helicase. In contrast, temperature does not seem to affect the IC₅₀ of the probe or its more potent analogues.

In conclusion, we have found that the commercial dyes thioflavine S (CID 415676) and primuline (CID 415713) contain potent compounds for the inhibition of the NS3 helicase of HCV. We show here that minor components of primuline (CID 415713) inhibit both the HCV helicase and HCV replicon replication. The antiviral potential of these trimeric and tetrameric benzothiazoles inspired the derivatization of the more abundant dimeric constituent. Several derivatives were found to be close in potency to the isolated trimer or tetramer in the MBHA and to possess improved DNA-binding profiles. Importantly, the antiviral potential of this class of helicase inhibitors does not appear to depend entirely on either their ability to inhibit HCV helicase or bind DNA. We speculate that the ability to inhibit HCV replication results from a compound’s ability to enter Huh7.5 cells, which can be monitored by examining compound fluorescence when they are administered to cells. In cell culture, both thioflavine S (CID 415676) and primuline (CID 415713) do not stain Huh7.5 cells, but preliminary data suggest that some of the derivatives enter cells. As described below, effects of the most potent replicon inhibitors of HCV replication (e.g.ML283) are presently being examined in more detail. The results of these studies will be used to inform additional chemistry efforts toward helicase inhibitors, which is an ongoing concern of our laboratories.

4.2. Mechanism of Action Studies

Experiments geared towards understanding how the probe inhibits the helicase reaction in vitro and how the probe disrupts cellular HCV replication are presently in progress in our laboratories.

Biochemical assays are being performed similar to the ones done with primuline, thioflavine S, and other helicase inhibitors (32). Briefly, steady state kinetics is being used to test if the probe competes with other known NS3 ligands like ATP, RNA, the NS4A peptide, and a variety of NS3 protease inhibitors. Intrinsic protein fluorescence and the fluorescence of the probe itself are being used to examine the direct interaction of the protein and probe, with Förster resonance energy transfer being used to estimate atomic distances. Some of the more soluble analogues are being used with isothermal titration calorimetry to better understand the thermodynamics explaining interaction between the probe, NS3 and nucleic acids.

Cellular experiments in progress include more detailed studies of the kinetics of HCV inhibition using both HCV replicons and cells infected with the JFH1 strain virus, which is uniquely capable of replication in cell culture. We are also examining methods to best deliver the probe in the cells to yield a more potent and reproducible antiviral effect. The probe and its more potent analogues are also being added to cells with HCV protease inhibitors and other drugs in clinical trials to examine for synergistic and antagonistic effects that might occur when helicase inhibitors are combined with other direct acting antivirals.

4.3. Planned Future Studies

The obvious next experiment to be done with this probe would be to test its effect on the replication of authentic HCV. All the work here was done using purified, recombinant HCV proteins and modified HCV subgenomic replicons. Such systems are HTS compatible, but they fail to recapitulate the complete HCV lifecycle. Future studies should therefore involve HCV capable of replicating in cell culture (47). We suspect that the probe will be more effective with authentic HCV because the helicase has been recently shown to interact with the HCV structural protein, core, which is not present in subgenomic replicons (48). The fact that the probe analogue most active in cells, CID 50930749, is less selective than the probe raises the interesting possibility that the antiviral effect seen with other helicase inhibitors is not due to a direct attack on NS3h but rather a non-specific attack on related helicase or nucleic acid binding proteins. This is an interesting hypothesis that could be rigorously tested using CID 50930749, the probe, and some of the other compounds reported here. Since probe solubility might still limit its use in such studies, future efforts will also be devoted to synthesizing and testing more soluble analogues.

In other studies, the probe and analogues will also be tested against RNA helicases encoded by other organisms, as well as a panel of ATPase and proteases. If the probe lacks specificity in such experiments, more analogues will be designed to enhance specificity. Planned future studies that could enhance specificity include more detailed structural studies using X-ray crystallography, followed by structure-based design of more specific derivatives of the probe.

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