30 research outputs found
Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis
SummaryBiotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts
Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis
SummaryBiotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts
Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors
Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KDs ≤ 2 nM. Additionally, we obtained high-resolution cocrystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhancement in accumulation of a C-2'-α analogue over the corresponding C-2'-β analogue, consistent with their differential whole-cell activity
Characterization of caspase‐2 inhibitors based on specific sites of caspase‐2‐mediated proteolysis
Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1–6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors
Fragment-Based Identification of an Inducible Binding Site on Cell Surface Receptor CD44 for the Design of Protein–Carbohydrate Interaction Inhibitors
Selective inhibitors
of hyaluronan (HA) binding to the cell surface
receptor CD44 will have value as probes of CD44-mediated signaling
and have potential as therapeutic agents in chronic inflammation,
cardiovascular disease, and cancer. Using biophysical binding assays,
fragment screening, and crystallographic characterization of complexes
with the CD44 HA binding domain, we have discovered an inducible pocket
adjacent to the HA binding groove into which small molecules may bind.
Iterations of fragment combination and structure-driven design have
allowed identification of a series of 1,2,3,4-tetrahydroisoquinolines
as the first nonglycosidic inhibitors of the CD44–HA interaction.
The affinity of these molecules for the CD44 HA binding domain parallels
their ability to interfere with CD44 binding to polymeric HA in vitro.
X-ray crystallographic complexes of lead compounds are described and
compared to a new complex with a short HA tetrasaccharide, to establish
the tetrahydroisoquinoline pharmacophore as an attractive starting
point for lead optimization
Searching for Likeness in a Database of Macromolecular Complexes
A software tool and workflow based
on distance geometry is presented
that can be used to search for local similarity in substructures in
a comprehensive database of experimentally derived macromolecular
structure. The method does not rely on fold annotation, specific secondary
structure assignments, or sequence homology and may be used to locate
compound substructures of multiple segments spanning different macromolecules
that share a queried backbone geometry. This generalized substructure
searching capability is intended to allow users to play an active
part in exploring the role specific substructures play in larger protein
domains, quaternary assemblies of proteins, and macromolecular complexes
of proteins and polynucleotides. The user may select any portion or
portions of an existing structure or complex to serve as a template
for searching, and other structures that share the same structural
features are identified, retrieved and overlaid to emphasize substructural
likeness. Matching structures may be compared using a variety of integrated
tools including molecular graphics for structure visualization and
matching substructure sequence logos. A number of examples are provided
that illustrate how generalized substructure searching may be used
to understand both the similarity, and individuality of specific macromolecular
structures. Web-based access to our substructure searching services
is freely available at https://drugsite.msi.umn.edu
Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)
Human
histidine triad nucleotide binding protein 1 (hHint1) is
classified as an efficient nucleoside phosphoramidase and acyl-adenosine
monophosphate hydrolase. Human Hint1 has been shown to be essential
for the metabolic activation of nucleotide antiviral pronucleotides
(i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir.
The active site of hHint1 comprises an ensemble of strictly conserved
histidines, including nucleophilic His112. To structurally investigate
the mechanism of hHint1 catalysis, we have designed and prepared nucleoside
thiophosphoramidate substrates that are able to capture the transiently
formed nucleotidylated-His112 intermediate (<b>E*</b>) using
time-dependent crystallography. Utilizing a catalytically inactive
hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme–substrate
(<b>ES</b><sup><b>1</b></sup>) and product (<b>EP</b><sup><b>2</b></sup>) complexes were also cocrystallized, respectively,
thus providing a structural map of the reaction trajectory. On the
basis of these observations and the mechanistic necessity of proton
transfers, proton inventory studies were carried out. Although we
cannot completely exclude the possibility of more than one proton
in flight, the results of these studies were consistent with the transfer
of a single proton during the formation of the intermediate. Interestingly,
structural analysis revealed that the critical proton transfers required
for intermediate formation and hydrolysis may be mediated by a conserved
active site water channel. Taken together, our results provide mechanistic
insights underpinning histidine nucleophilic catalysis in general
and hHint1 catalysis, in particular, thus aiding the design of future
proTides and the elucidation of the natural function of the Hint family
of enzymes