Replicon Assay Research Articles

First Example of Phosphoramidate Approach Applied to a 4‘-Substituted Purine Nucleoside (4‘-Azidoadenosine): Conversion of an Inactive Nucleoside to a Submicromolar Compound versus Hepatitis C Virus

We report on the synthesis of the anti hepatitis C virus (HCV) agent 4'-azidoadenosine (1) and the application of the phosphoramidate ProTide technology to this nucleoside. The synthesis of 1 was achieved through an epoxide intermediate followed by regio- and stereoselective ring opening by azidotrimethylsilane in the presence of a Lewis acid. Compound 1 did not inhibit HCV replication in cell culture at concentrations up to 0.1 mM. However, a submicromolar active agent could be derived from 1 by the application of the ProTide technology. All the phosphoramidates prepared were L-alanine derivatives with variations in the aryl moiety and in the ester part of the amino acid. The benzyl ester and the l-naphthyl phosphate (18) had the best activity in replicon assay. Phosphoramidates (18-21) achieved a significant improvement in antiviral potency over the parent nucleoside (1) with no increase in cytotoxicity.

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.

Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: Use of cyclopentane and cyclopentene P2-motifs

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4 R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K i value of 0.41 nM and an EC 50 value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.

5′- O-Masked 2′-deoxyadenosine analogues as lead compounds for hepatitis C virus (HCV) therapeutic agents

On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus, a series of nucleoside analogues whose 5′-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and ether were synthesized and evaluated to develop novel anti-hepatitis C virus (HCV) agents. Among these, several 5′- O-masked analogues of 6-chloropurine-2′-deoxyriboside (e.g., 5′- O-benzoyl, 5′- O- p-methoxybenzoyl, and 5′- O-benzyl analogues) were found to exhibit effective anti-HCV activity. In particular, the 5′- O-benzoyl analogue exhibited the highest potency with an EC 50 of 6.1 μM in a cell-based HCV replicon assay. Since the 5′- O-unmasked analogue (i.e., 6-chloropurine-2′-deoxyriboside) was not sufficiently potent (EC 50 = 47.2 μM), masking of the 5′-hydroxyl group seems to be an effective method for the development of anti-HCV agents. Presently, we hypothesize two roles for the 5′- O-masked analogues: One is the role as an anti-HCV agent by itself, and the other is as a prodrug of its 5′- O-demasked (deprotected) derivative.

Identification of novel epoxide inhibitors of hepatitis C virus replication using a high-throughput screen.

Using our high-throughput hepatitis C replicon assay to screen a library of over 8,000 novel diversity-oriented synthesis (DOS) compounds, we identified several novel compounds that regulate hepatitis C virus (HCV) replication, including two libraries of epoxides that inhibit HCV replication (best 50% effective concentration, < 0.5 microM). We then synthesized an analog of these compounds with optimized activity.

Allosteric Inhibitors of Hepatitis C Polymerase: Discovery of Potent and Orally Bioavailable Carbon-Linked Dihydropyrones

The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.

Liver-Targeted Prodrugs of 2‘-C-Methyladenosine for Therapy of Hepatitis C Virus Infection

2'-C-Methyladenosine exhibits impressive inhibitory activity in the cell-based hepatitis C virus (HCV) subgenomic replicon assay, by virtue of intracellular conversion to the corresponding nucleoside triphosphate (NTP) and inhibition of NS5B RNA-dependent RNA polymerase (RdRp). However, rapid degradation by adenosine deaminase (ADA) limits its overall therapeutic potential. To reduce ADA-mediated deamination, we prepared cyclic 1-aryl-1,3-propanyl prodrugs of the corresponding nucleoside monophosphate (NMP), anticipating cytochrome P450 3A-mediated oxidative cleavage to the NMP in hepatocytes. Lead compounds identified in a primary rat hepatocyte screen were shown to result in liver levels of NTP predictive of efficacy after intravenous dosing to rats. The oral bioavailability of the initial lead was below 5%; therefore, additional analogues were synthesized and screened for liver NTP levels after oral administration to rats. Addition of a 2',3'-carbonate prodrug moiety proved to be a successful strategy, and the 1-(4-pyridyl)-1,3-propanyl prodrug containing a 2',3'-carbonate moiety displayed oral bioavailability of 39%.

Highlights of the 20th International Conference on Antiviral Research (ICAR)

The International Conference on Antiviral Research (ICAR) meetings cover all aspects of antiviral research into human viruses. The meeting included two major award lectures, invited plenary speakers and a minisymposium, based this year on HCV. In addition, there were many contributor presentations, both oral and poster. This report focuses on the major highlights of the contributor presentations. A full report, including summaries of all the invited speakers and the pre-conference clinical update satellite symposium, will be included in the International Society of Antiviral Research (ISAR) news to be published in Antiviral Chemistry and Chemotherapy (18.4).

Discovery of novel dialkyl substituted thiophene inhibitors of HCV by in silico screening of the NS5B RdRp

A novel 5,4-dialkyl substituted thiophene was discovered by in silico screening of the 3D polymerase crystal structure (1GX6) that demonstrated single digit micromolar HCV inhibition activity in the replicon assay and dose-dependent inhibition in the replicase complex assay. Subsequently, SAR was explored with a small set of dialkyl and tetrahydro-benzo thiophenes. Since these thiophenes inhibit synthesis of both, single- and double-stranded RNAs, their mechanism of action is distinct from other known HCV inhibitors.

Breaking pseudo-twofold symmetry in the poliovirus 3'-UTR Y-stem by restoring Watson-Crick base pairs

The previously described NMR structure of a 5'-CU-3'/5'-UU-3' motif, which is highly conserved within the 3'-UTR Y-stem of poliovirus-like enteroviruses, revealed striking regularities of the local helix geometry, thus retaining the pseudo-twofold symmetry of the RNA helix. A mutant virus with both pyrimidine base pairs changed into Watson-Crick replicated as wild type, indicating the functional importance of this symmetry relation in viral RNA replication. Here we investigated the effect of changing only one of the two pyrimidine base pairs to Watson-Crick. We determined the NMR structures of two Y-stem variants: one containing the 5'-CU-3'/5'-AU-3' motif, which has been found in wild-type virus isolates as well, and the other containing a 5'-CU-3'/5'-UG-3' motif, which is not present in any enterovirus sequenced to date. Both structures show single pyrimidine mismatches with intercalated bases. In the 5'-CU-3'/5'-AU-3' motif a C-U Watson-Crick-type base pair is formed that retains the pseudo-twofold symmetry, while in the 5'-CU-3'/5'-UG-3' motif a single asymmetric U-U mismatch breaks the twofold symmetry. Surprisingly, for the nonnatural variant no effect of the single base-pair replacement was observed on polioviral RNA replication using an in vitro replicon assay.

Chemical and biological effects of substitution of the 2-position of ring-expanded (‘fat’) nucleosides containing the imidazo[4,5- e][1,3]diazepine-4,8-dione ring system: The role of electronic and steric factors on glycosidic bond stability and anti-HCV activity

The attempted removal of the aralkyl group of 2-bromo-1- p-methoxybenzyl-6-octylimidazo[4,5- e][1,3]diazepine ( ZP- 33) with trifluoroacetic acid resulted in replacement of the bromo group with a carbonyl at position-2 in addition to the desired deprotection at the 1-position. 2′-Deoxynucleosides of 2-bromo-substituted-imidazole-4,5-diesters ( ZP- 35 and ZP- 103) were synthesized by direct glycosylation of the corresponding heterocycles. The attempted ring-closure of the above nucleosides resulted in deglycosylation to form the starting heterocycles. The 2-phenyl derivatives of the above nucleosides ( ZP- 45 and ZP- 73) were successfully prepared by Suzuki coupling with the appropriate phenylboronic acids, but the attempted ring-closure with guanidines to form the desired 5,7-fused ring-expanded nucleosides (RENs) resulted once again in the formation of the corresponding heterocyclic aglycons ( ZP- 64 and ZP- 75). The first successful 2-substituted REN ( ZP- 110) was synthesized by replacing the 2-deoxyribose sugar moiety with a ribosyl group and replacing the bromo group with a p-methoxyphenyl substituent at the 2-position. A number of imidazole riboside diester precursors containing a substituted phenyl group at the 2-position were synthesized in order to prepare analogues of ZP- 110. The substituents on the phenyl ring included a variety of electron-donating or electron-withdrawing groups operating through inductive and/or resonance effects. However, the final ring-closure of the diesters with guanidines in order to prepare RENs was successful only in a limited number of cases, including the ones containing a p-fluorophenyl ( ZP- 121), a m-methoxyphenyl ( ZP- 122), or an unsubstituted phenyl ( NZ- 53) at the 2-position. Deglycosylation and incomplete ring-closure of the intermediates were the major problems encountered with most other cases. The stability of glycosidic bonds was found to be dependent on several factors including, but not limited to, the electron-donating inductive effect of the 2-phenyl substituents and the temperature of the reaction medium. The three target RENs ZP- 110, ZP- 121, and ZP- 122 were screened for in vitro anti-HCV activity, employing an HCV RNA replicon assay. While ZP- 121 was inactive, the other two compounds showed only weak anti-HCV activity.

590] INHIBITION OF HCV REPLICATION BY PSI-6130: MECHANISM OF BIOCHEMICAL ACTIVATION AND INHIBITION

Background: Nearly 2% of the U S . population and an estimated 170 million people worldwide are HCV carriers. The current standard of care, a combination of pegylated interferon and ribavirin, has limited efficacy. We have identified a nucleoside analog, (3-D-2’-deoxy-2’-fluoro2’-C-methylcytidine (PSI-61 30), which is a specific, potent and noncytotoxic inhibitor of HCV in the subgenomic replicon assay. To inhibit the HCV NS5B polymerase, PSI-6 130 must be phosphorylated to the 5’triphosphate. The enzymes involved in the phosphorylation of PSI-61 30 and inhibition of HCV NS5B were investigated. Methods: Anti-HCV activity was measured using subgenomic-HCV replicon cells and a real-time PCR assay. Spectrophotometric assays and recombinant deoxycytidine kinase, uridine-cytidine kinase, UMP-CMP kinase, and nucleoside diphosphate kinase (NDPK) were used to investigate the phosphorylation pathway of PSI-6130 to the corresponding 5’-triphosphate. The NS5B RNA polymerase reaction was performed in the presence of PSI-6 130-TP or other 2’-C-Me-nucleoside triphosphates using 32P-labeled UTP and the amount of radioactivity in the product was measured. Results: An EC90 (concentration producing I loglo reduction in viral RNA) value of 4.6f2.0pM was determined for PSI-6130 and was essentially inactive against other viruses including BVDV Little or no cytotoxicity or mitochondrial toxicity was observed with cells treated with PSI-6 130. Purified dCK, UMP-CMP kinase, and NDPK phosphorylated PSI-61 30 to the 5’-mono-, di-, and triphosphate derivatives respectively. PSI-6130-TP was an alternative substrate inhibitor of purified NSSB. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B resulted in chain-termination. The steady-state inhibition constant for PSI6 130-TP was Ki = 4.3 pM. Against the S282T NS5B enzyme PSI-6 130-TP showed only a 3-fold resistance compared to a 12-fold resistance for 2‘C-Me-cytidine-TP and a 150-fold resistance for 2’-C-Me-adenosine-TP. Conclusion: PSI-6 130 demonstrated potent and specific anti-HCV activity in cell-based assays with little or no cytotoxicity and mitochondrial toxicity. PSI-6 130 was efficiently phosphorylated to its active 5’-triphosphate form. Deoxycytidine kinase, UMP-CMP kinase, and NDPK were identified as enzymes involve in the phosphorylation pathway of PSI-6130. PSI6130-TP inhibited HCV NSSB RNA polymerase and acted as a chainterminator upon incorporation into the elongating RNA strand by HCV NS5B.

Dynamic Hepatitis C Virus Genotypic and Phenotypic Changes in Patients Treated With the Protease Inhibitor Telaprevir

Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur. A highly sensitive sequencing method was developed in which approximately 80 clones/sample were analyzed to identify mutations in the NS3 protease catalytic domain in HCV genotype-1-infected patients dosed with 450 mg every 8 hours, 750 mg every 8 hours, or 1250 mg every 12 hours of telaprevir for 14 days. Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36+155, 36+156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Changes in the frequency of mutations after the end of dosing showed an inverse relationship between in vivo viral fitness and resistance. In the absence of telaprevir selective pressure the majority of resistant variants were replaced by wild-type virus within 3-7 months. Resistant HCV isolates are selected rapidly during therapy with the highly active protease inhibitor telaprevir. Combination therapy with pegylated interferon-alfa or other direct antiviral drugs seem mandatory to avoid developing resistance.

Discovery of Conformationally Constrained Tetracyclic Compounds as Potent Hepatitis C Virus NS5B RNA Polymerase Inhibitors

We report a new series of hepatitis C virus NS5B RNA polymerase inhibitors containing a conformationally constrained tetracyclic scaffold. SAR studies led to the identification of 6,7-dihydro-5H-benzo[5,6][1,4]diazepino[7,1-a]indoles (19 and 20) bearing a basic pendent group with high biochemical and cellular potencies. These compounds displayed a very small shift in cellular potency when the replicon assay was performed in the presence of human serum albumin.

Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

Synthesis of pyrrolo[2,3- d]pyrimidine nucleoside derivatives as potential anti-HCV agents

Several Toyocamycin ( 4) analogues were examined for their ability to inhibit HCV RNA in a replicon assay. Among the compounds examined 4-methylthio ( 18) and 5-carboxamide oxime derivatives ( 23 and 27) of Toyocamycin were found to have good activity and selectivity.

Cytostatic and antiviral 6-arylpurine ribonucleosides. Part 7: Synthesis and evaluation of 6-substituted purine l-ribonucleosides

A series of purine l-ribonucleosides 2a– 2i bearing diverse C-substituents (alkyl, aryl, hetaryl or hydroxymethyl) in the position 6 were prepared by Pd-catalyzed cross-coupling reactions of 6-chloro-9-(2,3,5-tri- O-acetyl-β- l-ribofuranosyl)purine with the corresponding organometallics followed by deprotection. Unlike their d-ribonucleoside enantiomers that possess strong cytostatic and anti-HCV activity, the l-ribonucleosides were inactive except for 6-benzylpurine nucleoside 2h showing moderate anti-HCV effect in replicon assay. A triphosphate of 2h did not inhibit HCV RNA polymerase.

Inhibition of Hepatitis C Replicon RNA Synthesis by β-D-2′-deoxy-2′-fluoro-2′-C-Methylcytidine: A Specific Inhibitor of Hepatitis C Virus Replication

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.

Nucleoside Analog Inhibitors of Hepatitis C Virus Replication

Of the 30 compounds currently marketed in the United States for treatment of viral infections, 15 are nucleoside analogs, demonstrating the utility of this class of compound as a source of antiviral drugs. The success of nucleoside analogs in treating other viral infections provides a compelling rationale for the significant effort that is currently being devoted to the discovery and development of nucleoside analogs to treat infection by hepatitis C virus (HCV) that may lead to improvements in response rates compared to currently available therapies. Several different approaches have been adopted to identify promising analogs, including the use of surrogate viruses in cell culture assays, screening in the cell-based bicistronic HCV replicon assay, and screening nucleoside triphosphates for the ability to inhibit the activity of the HCV RNA-dependent RNA polymerase in vitro. Several classes of ribonucleoside analogs with modifications of the ribose inhibit HCV replication. Nucleoside analogs incorporating a 2'-C-methyl modification are potent inhibitors in the replicon assay in the absence of cytotoxicity, and appear to exert their inhibition by acting as functional chain terminators of RNA synthesis. NM283, a prodrug of 2'-C-methylcytidine, has entered clinical trials and demonstrated viral load reductions in subjects infected with genotype 1 HCV, a genotype known to be difficult to treat effectively with currently approved therapies. Overall, results to date offer encouragement that improved therapies to treat HCV infection including newly developed nucleoside analogs may become available within the next few years.

Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.