ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Benzamides/pharmacology , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Kinesins/genetics , Kinesins/metabolism , Nucleotides/metabolism , Quinazolines/pharmacology , Thermodynamics , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Substitution , Benzamides/metabolism , Biocatalysis , Calorimetry , Circular Dichroism , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/metabolism , Humans , Kinesins/antagonists & inhibitors , Kinetics , Magnesium/chemistry , Magnesium/metabolism , Models, Molecular , Nucleotides/chemistry , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Pyrimidines/chemistry , Pyrimidines/metabolism , Quinazolines/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Temperature , Thiones/chemistry , Thiones/metabolism , Transition TemperatureABSTRACT
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.
Subject(s)
HIV Integrase Inhibitors/isolation & purification , HIV Integrase Inhibitors/pharmacology , HIV Integrase/drug effects , Intercellular Signaling Peptides and Proteins/metabolism , Base Sequence , DNA Primers , Drug Evaluation, Preclinical , HIV Integrase/metabolism , HumansABSTRACT
The structures of both native and S139A holo-HCV NS3/4A protease domain 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 contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. 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, which is currently in clinical trials.
Subject(s)
Hepacivirus/enzymology , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Models, Molecular , Molecular Structure , Proline/chemistryABSTRACT
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.
Subject(s)
Antiviral Agents/chemical synthesis , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Hepacivirus/enzymology , Proline/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Antiviral Agents/chemistry , Binding Sites , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins , Models, Molecular , Proline/chemical synthesis , Proline/chemistry , Protein Conformation , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Prolonged hepatitis C infection is the leading cause for cirrhosis of the liver and hepatocellular carcinoma. The etiological agent HCV virus codes a single polyprotein of approximately 3000 amino acids that is processed with the help of a serine protease NS3A to produce structural and non-structural proteins required for viral replication. Inhibition of NS3 protease can potentially be used to develop drugs for treatment of HCV infections. Herein, we report the development of a series of novel NS3 serine protease inhibitors derived from 2-aza-bicyclo[2.2.1]-heptane carboxylic acid with potential therapeutic use for treatment of HCV infections.
Subject(s)
Bridged Bicyclo Compounds , Hepacivirus/drug effects , Replicon/drug effects , Serine Proteinase Inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Binding Sites , Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/chemistry , Bridged Bicyclo Compounds/pharmacology , Hepacivirus/chemistry , Hepacivirus/enzymology , Molecular Structure , Protein Binding , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , X-Ray DiffractionABSTRACT
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.
