Significant Differences in Capsid Properties and Potency Between Adeno-Associated Virus Vectors Produced in Sf9 and HEK293 Cells.

For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.

P4 capped amides and lactams as HCV NS3 protease inhibitors with improved potency and DMPK profile.

SAR studies on the extension of P3 unit of Boceprevir (1, SCH 503034) with amides and lactams and their synthesis is described. Extensive SAR studies resulted in the identification of 36 bearing 4, 4-dimethyl lactam as the new P4 cap unit with improved potency (K(i)( *)=15nM, EC 90=70nM) and pharmacokinetic properties (Rat AUC (PO)=3.52microMh) compared to 1.

The introduction of P4 substituted 1-methylcyclohexyl groups into Boceprevir: a change in direction in the search for a second generation HCV NS3 protease inhibitor.

In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.

Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

Potent inhibitors of HCV-NS3 protease derived from boronic acids.

Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

Discovery of novel P3 sulfonamide-capped inhibitors of HCV NS3 protease. Inhibitors with improved cellular potencies.

Hepatitis C Virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 200 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or PEG-interferon alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only approximately 50% of the patients showing sustained virological response. We recently disclosed the discovery of Boceprevir, SCH 503034 (1), which is a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been shown to be efficacious in humans and is currently undergoing clinical trials. As second generation compounds, we have further explored various novel structures with the aim of improving enzyme and cellular binding activities of 1. Herein, we disclose our efforts toward the identification of a novel P(3) sulfonamide-capped inhibitor that demonstrated improved binding and cellular activity compared to 1. X-ray structure of one of these inhibitors bound to the enzyme revealed a hydrogen bond of the P(3) sulfonamide group to Cys-159 which resulted in improved binding and cellular potency.

Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay.

Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.

Characterization of resistance mutations against HCV ketoamide protease inhibitors.

An issue of clinical importance in the development of new antivirals for HCV is emergence of resistance. Several resistance loci to ketoamide inhibitors of the NS3/4A protease have been identified (residues V36, T54, R155, A156, and V170) by replicon and clinical studies. Using SCH 567312, a more potent protease inhibitor derived from SCH 503034 (boceprevir) series, we identified two new positions (Q41 and F43) that confer resistance to the ketoamide class. The catalytic efficiency of protease enzymes was not affected by most resistance mutations, whereas replicon fitness varied with specific mutations. SCH 503034 and another ketoamide inhibitor, VX-950 (telaprevir), showed moderate losses of activity against most resistance mutations (< or =10-fold); the highest resistance level was conferred by mutations at A156 locus. Although SCH 503034 and VX-950 bind similarly to the active site, differences in resistance level were observed with specific mutations. Changes at V36 and R155 had more severe impact on VX-950, whereas mutations at Q41, F43 and V170 conferred higher resistance to SCH 503034. Structural analysis of resistance mutations on inhibitor binding is discussed.

Discovery of the HCV NS3/4A protease inhibitor (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-3- [2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (Sch 503034) II. Key steps in structure-based optimization.

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.

Application of ring-closing metathesis for the synthesis of macrocyclic peptidomimetics as inhibitors of HCV NS3 protease.

An efficient synthetic approach for the preparation of macrocyclic peptidomimetics for inhibition of HCV NS3 is presented. The macrocyclic core is built using ring-closing metathesis (RCM) of a tripeptidic diene. The presented approach allows the introduction of heteroatoms in strategic places along the macrocyclic ring. The methyl ester moiety in the RCM products was synthetically manipulated to install a keto-amide moiety via a Passerini reaction.

Potent 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-based macrocyclic inhibitors of hepatitis C virus NS3 protease.

The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are alpha-ketoamide-type HCV serine protease inhibitors with modest potency. We envisioned that the 1,2,3,4-tetrahydroisoquinoline-3-carboxylamide (Tic) moiety could be cyclized to the P3 capping group. The resulting macrocycle could enhance the binding through its extra contact with the Ala156 methyl group. Macrocyclization could also provide a less peptidic HCV inhibitor. Synthesis started from dipeptide 5, which was obtained via a coupling of two amino acid derivatives. The N-terminal was capped as hept-6-enoylamide to give 6. Hydroboration of the double bond afforded alcohol 7, the precursor to the macrocycle 8. The macrocyclization was achieved under Mitsunobu conditions (PPh(3), ADDP). The macrocyclic acid 9 was then combined with appropriate right-hand fragments 12, 14, or 16, which was prepared from common intermediate 11. Finally, oxidation of alpha-hydroxyamide provided target molecule alpha-ketoamides 17, 18, and 21. The C-terminal esters were then elaborated to carboxylic acids 19 and 20, and amides 20 and 23. The inhibitors 17-23 were tested in HCV NS3 protease continuous assay. Tripeptide 17 was more potent than the larger acyclic tetrapeptide 1. The tetrapeptides 18-20 were as active as 17. Most significantly, the pentapeptides (21-23) were much better inhibitors (K(i) = 0.015-0.26 microM). The carboxylic acid (22) and amide (23) were 57-80 times more potent than the acyclic analogue 2. The X-ray crystal structure of compound 23 bound to the protease revealed that the macrocycle adopted a donutlike conformation and had close contact with the Ala156 methyl group. The ketone carbonyl formed a reversible covalent bond with Ser139. The n-propyl of P1 novaline and the aromatic ring of P2' phenylglycine formed a C-shaped clamp around the Lys136 side chain.

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.

Discovery of SCH446211 (SCH6): a new ketoamide inhibitor of the HCV NS3 serine protease and HCV subgenomic RNA replication.

Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

Discovery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a selective, potent, orally bioavailable hepatitis C virus NS3 protease inhibitor: a potential therapeutic agent for the treatment of hepatitis C infection.

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.

Design and synthesis of depeptidized macrocyclic inhibitors of hepatitis C NS3-4A protease using structure-based drug design.

Hepatitis C virus (HCV) NS3, when bound to NS-4A cofactor, facilitates development of mature virons by catalyzing cleavage of a polyprotein to form functional and structural proteins of HCV. The enzyme has a shallow binding pocket at the catalytic site, making development of inhibitors difficult. We have designed, preorganized, and depeptidized macrocyclic inhibitors from P(4) to P(2)' and optimized binding to 0.1 microM. The structure of an inhibitor bound to the enzyme was also solved.

Design, synthesis, and biological activity of m-tyrosine-based 16- and 17-membered macrocyclic inhibitors of hepatitis C virus NS3 serine protease.

The limited efficacy and considerable side effects of currently available therapies for the treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the development of safe and effective new therapeutics. The pentapeptide alpha-ketoamides of type 1 were weak HCV inhibitors with a binding constant, Ki, above 5 microM. We envisioned that cyclization of a P2 phenyl side chain to a P3 capping group could enhance binding through an interaction of the resulting macrocycle with the methyl group of Ala156 on the enzyme backbone. The macrocyclic dipeptide moiety would also decrease the peptidic nature of the inhibitors. The synthesis of macrocyclic HCV inhibitors started from m-tyrosine methyl ester. Two consecutive couplings, first, with Boc-cyclohexylglycine and, then, with hept-6-enoic acid, provided compound 6. The alkene was converted to an alcohol via hydroboration. The key macrocyclization of phenol alcohol 7 was achieved through a Mitsunobu reaction. Both 16- and 17-membered macrocycles (8 and 21) were prepared. After hydrolysis, the macrocyclic acids (15 and 22) were coupled to the right-hand tripeptide (14) to afford alpha-hydroxyamides, which upon Dess-Martin periodinane oxidation furnished the desired alpha-ketoamides. Esters, acids, and amides were incorporated at the C-terminal of these peptides. These inhibitors were tested in an HCV protease continuous assay. The binding constants (Ki) indicated that the 16-membered macrocyclic inhibitors (23 and 24) were less potent than the 17-membered analogues (16-19). It was also evident that C-terminal acids (i.e., 17) and amides (18 and 19) (Ki range: 0.16-0.31 microM) were much better inhibitors than tert-butyl esters (16 and 23). The X-ray crystal structure of compound 17 bound to the enzyme revealed that the macrocycle formed a "donut"-shaped ring around the methyl group of Ala156. P2' phenyl and P1 propyl groups wrapped around the Lys136 side chain, forming a "C"-shaped clamp. The 17-membered macrocyclic inhibitors 17-19 were significantly more potent than the acyclic pentapeptide 1.

Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin.

Recent successes of adeno-associated virus (AAV)-based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE), we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering) differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37) and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9). The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA) resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors' in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

Application of the lambda-dynamics method to evaluate the relative binding free energies of inhibitors to HCV protease.

The lambda-dynamics method was used to calculate the relative binding free energies of inhibitors to the hepatitis C virus (HCV) protease. A total of seven HCV protease p-side product inhibitors were used in this study. The inhibitors are 6-mer peptides spanning P6-P1 (Ac-Asp-d-Glu-Leu-Ile-Cha-P1-CO(2)H). For this protein, S1 is a major hydrophobic pocket for binding. Binding of various residues to this pocket was investigated through free energy simulations and experimental inhibition constants. Several 300 ps lambda-dynamics simulations in explicit solvent were performed. The relative binding free energy was estimated from these simulations. From a single simulation, the inhibitors can be correctly classified into highly potent and weakly potent groups. The multiple simulations give an accurate rank ordering of inhibitor potency; computed and experimental binding free energies agree with 0.6 kcal/mol for five of the seven inhibitors. In addition, free energy perturbation (FEP) calculations were carried out to validate the results from lambda-dynamics. A total of 6 ligand pairs were compared. For each pair, 5-11 windows were used to map one ligand to the other. The cumulative simulation time was over 2 ns for each ligand pair. For four of the six ligand pairs, the lambda-dynamics free energy difference fits better than the FEP difference to the experimental value. The fact that the lambda-dynamics method achieved similar results in only a fraction of the total simulation time for FEP further demonstrates the robustness of the lambda-dynamics method.

Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor.

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.