Discovery of a Distinct Chemical and Mechanistic Class of Allosteric HIV-1 Integrase Inhibitors with Antiretroviral Activity.

Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.

Chronic inhibition of 11 ß -hydroxysteroid dehydrogenase type 1 activity decreases hypertension, insulin resistance, and hypertriglyceridemia in metabolic syndrome.

Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11 ß -hydroxysteroid dehydrogenase type 1 (11 ß -HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11 ß -HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11 ß -HSD1. Compound 11 significantly decreased 11 ß -HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11 ß -HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.

Development of a high-throughput cell-based assay for 11beta-hydroxysteroid dehydrogenase type 1 using BacMam technology.

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA.

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

Mimicking damaged DNA with a small molecule inhibitor of human UNG2.

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs.

Uracil DNA glycosylase (UNG) locates uracil and its structural congener thymine in the context of duplex DNA using a base flipping mechanism. NMR imino proton exchange measurements were performed on free and UNG-bound DNA duplexes in which a single thymine (T) was paired with a series of adenine analogues (X) capable of forming one, two, or three hydrogen bonds. The base pair opening equilibrium for the free DNA increased 55-fold as the number of hydrogen bonds decreased, but the opening rate constants were nearly the same in the absence and presence of UNG. In contrast, UNG was found to slow the base pair closing rate constants (kcl) compared to each free duplex by a factor of 3- to 23-fold. These findings indicate that regardless of the inherent thermodynamic stability of the TX pair, UNG does not alter the spontaneous opening rate. Instead, the enzyme holds the spontaneously expelled thymine (or uracil) in a transient extrahelical sieving site where it may partition forward into the enzyme active site (uracil) or back into the DNA base stack (thymine).

Synthesis and high-throughput evaluation of triskelion uracil libraries for inhibition of human dUTPase and UNG2.

Human nuclear uracil DNA glycosylase (UNG2) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA. These enzymes are desirable targets for small molecule inhibitors given their roles in a wide range of biological processes ranging from chromosomal rearrangements that lead to cancer, viral DNA replication, and the formation of toxic DNA strand breaks during anticancer drug therapy. To accelerate the discovery of such inhibitors, we have developed a high-throughput approach for directed library synthesis and screening. In this efficient technology, a uracil-aldehyde ligand is covalently tethered to one position of a trivalent alkyloxyamine linker via an oxime linkage, and then the vacant linker positions are derivatized with a library of aldehydes. The resulting triskelion oximes were directly screened for inhibitory activity and the most potent of these showed micromolar binding affinities to UNG2 and dUTPase.

Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development.

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases.

The origins of high-affinity enzyme binding to an extrahelical DNA base.

Base flipping is a highly conserved strategy used by enzymes to gain catalytic access to DNA bases that would otherwise be sequestered in the duplex structure. A classic example is the DNA repair enzyme uracil DNA glycosylase (UDG) which recognizes and excises unwanted uracil bases from DNA using a flipping mechanism. Previous work has suggested that enzymatic base flipping begins with dynamic breathing motions of the enzyme-bound DNA substrate, and then, only very late during the reaction trajectory do strong specific interactions with the extrahelical uracil occur. Here we report that UDG kinetically and thermodynamically prefers substrate sites where the uracil is paired with an unnatural adenine analogue that lacks any Watson-Crick hydrogen-bonding groups. The magnitude of the preference is a striking 43000-fold as compared to an adenine analogue that forms three H-bonds. Transient kinetic and fluorescence measurements suggest that preferential recognition of uracil in the context of a series of incrementally destabilized base pairs arises from two distinct effects: weak or absent hydrogen bonding, which thermodynamically assists extrusion, and, most importantly, increased flexibility of the site which facilitates DNA bending during base flipping. A coupled, stepwise reaction coordinate is implicated in which DNA bending precedes base pair rupture and flipping.

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.

3-Methyladenine DNA glycosylase I is an unexpected helix-hairpin-helix superfamily member.

The Escherichia coli enzyme 3-methyladenine DNA glycosylase I (TAG) hydrolyzes the glycosidic bond of 3-methyladenine (3-MeA) in DNA and is found in many bacteria and some higher eukaryotes. TAG shows little primary sequence identity with members of the well-known helix-hairpin-helix (HhH) superfamily of DNA repair glycosylases, which consists of AlkA, EndoIII, MutY and hOGG1. Unexpectedly, the three-dimensional solution structure reported here reveals TAG as member of this superfamily. The restricted specificity of TAG for 3-MeA bases probably arises from its unique aromatic rich 3-MeA binding pocket and the absence of a catalytic aspartate that is present in all other HhH family members.

Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme.

3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D-arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate. All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli, were believed not to require a metal cofactor for catalytic activity. However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme. Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes. One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori. In order to test this model, we have cloned the kdsa gene encoding H. pylori KDO-8-P synthase, and overexpressed and purified the protein. This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity. The Zn(2+) in the enzyme could be quantitatively replaced by Cd(2+), which increased the observed k(cat) by approximately 2-fold, and decreased the apparent K(m)(A-5-P) by approximately 6.5-fold. Furthermore, removal of the Zn(2+) from the enzyme did not greatly perturb its circular dichroism spectra. Thus, the divalent metal most likely serves as cofactor directly involved in catalysis.