HPSE Active

What is HPSE and what is its primary function in normal physiology?

Heparanase-1 (HPSE) is the only known mammalian enzyme responsible for the cleavage of heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs). It plays a key role in the remodeling and degradation of the extracellular matrix (ECM) . Under normal physiological conditions, HPSE expression is tightly regulated and contributes to wound healing, embryonic development, and immune response. Its endo-β-D-glucuronidase activity specifically cleaves the β(1,4)-glycosidic bonds between glucuronic acid and glucosamine residues in HS chains .

What are the validated methods for measuring HPSE enzymatic activity in experimental settings?

Several methodological approaches have been developed to measure HPSE activity:

• Radiolabeled substrate assays: This traditional method uses [³H]-labeled HS as substrate, with activity quantified by measuring radioactivity of [³H]-HS that remains bound to chicken histidine glycoprotein (cHRG) column after HPSE cleavage .

• Immobilized substrate methods: For higher throughput, [³H]-labeled HS can be immobilized on sepharose beads or streptavidin-coated plates, allowing for detection of HPSE activity through measurement of released radioactivity .

• Colorimetric-based assays: These methods rely on the specific binding of basic fibroblast growth factor (bFGF) to HS. HPSE activity is determined by measuring the reduction in bFGF binding to HS, as indicated by color development produced by TMB peroxidase .

How can researchers distinguish between active and latent forms of HPSE?

HPSE is synthesized as a latent 65 kDa proenzyme that requires proteolytic processing to generate the active heterodimer. The active form consists of an 8 kDa subunit (Gln36–Glu109) and a 50 kDa subunit (Lys158–Ile543) that associate non-covalently .

Researchers can distinguish between these forms through:

• SDS-PAGE and western blotting with antibodies specific to different domains

• Size exclusion chromatography separation based on molecular weight differences

• Activity assays, as only the processed heterodimeric form exhibits enzymatic activity

• Analysis of glycosylation patterns, as both forms may have distinct post-translational modifications

What computational design strategies have successfully improved HPSE stability for experimental applications?

The Protein Repair One Stop Shop (PROSS) algorithm has been successfully employed to design stable HPSE variants for bacterial expression. Unlike conventional consensus mutagenesis, PROSS combines phylogenetic sequence information with computational modeling using Rosetta forcefield-based approaches .

The implementation process involves:

• Submitting the crystal structure (e.g., PDB ID: 5E9C) to the PROSS server with constraints on residues involved in substrate binding and heterodimer interface

• Testing multiple design variants (typically 7 or more) with accumulated mutations

• Identifying the most soluble design through expression trials

In one successful application, a HPSE variant containing 26 amino acid substitutions (HPSE P6) maintained comparable catalytic activity and identical inhibition profiles to wild-type HPSE while dramatically improving solubility and expression yield (4 mg/L in E. coli) .

How do structure-activity relationships inform the design of small-molecule HPSE inhibitors?

Structure-activity relationship (SAR) studies have revealed critical insights for designing effective small-molecule HPSE inhibitors:

• Core structure importance: Several promising scaffolds have been identified through high-throughput screening, including benzimidazole cores, isoindole-5-carboxylic acid moieties, and benzoxazole compounds .

• Essential functional groups: Carboxylic acid groups are often critical for HPSE inhibition activity and cannot be modified without significant activity reduction .

• Optimization pathways: Systematic modification of:

  • Linker groups between core structures

  • Substitution patterns on pendant benzyl rings

  • Introduction of symmetric elements (e.g., urea compounds)

Key examples include compound series with benzimidazole cores where optimization of the linker groups improved both anti-HPSE activity and pharmacokinetic profiles. For instance, a compound designated as 3f demonstrated both good HPSE inhibitory activity and favorable pharmacokinetic properties in mice, while compound 3d showed the best anti-HPSE activity with an IC₅₀ of 230 nM .

What are the challenges in expressing active HPSE in prokaryotic systems and how can they be overcome?

Expressing active human HPSE in prokaryotic systems presents several challenges:

• Insolubility issues: Wild-type HPSE tends to form inclusion bodies when expressed in E. coli, particularly the 50 kDa subunit which typically remains completely insoluble .

• Post-translational modifications: HPSE contains glycosylation sites and disulfide bonds that are difficult to replicate in bacterial systems .

• Proper heterodimer formation: The active enzyme requires correct association of the 8 kDa and 50 kDa subunits.

These challenges can be overcome through:

• Computational redesign: Using PROSS algorithm to introduce stability-enhancing mutations while preserving function .

• Specialized expression strains: Employing E. coli Shuffle T7 Express cells that allow disulfide bond formation in the cytoplasm .

• Co-expression with chaperones: Trigger factor and GroEL/GroES chaperones help with proper protein folding .

• Dual expression vectors: Using vectors like pETDuet-1 for coordinated expression of both subunits .

• Optimized purification: Sequential purification using Ni²⁺-NTA, heparin, and size exclusion chromatography to obtain pure, homogeneous, heterodimeric HPSE .

How do small-molecule HPSE inhibitors differ mechanistically from heparan sulfate mimetics?

Small-molecule HPSE inhibitors and heparan sulfate (HS) mimetics differ significantly in their inhibitory mechanisms and pharmacological properties:

• Binding mechanism:

  • HS mimetics typically act as competitive substrate analogs that bind to the active site with high affinity

  • Small-molecule inhibitors often interact with specific residues within or adjacent to the active site through more defined molecular interactions

• Pharmacokinetic properties:

  • HS mimetics generally suffer from poor bioavailability and unfavorable pharmacokinetics

  • Small-molecule inhibitors typically have better drug-like characteristics, including improved bioavailability and tissue penetration

• Target selectivity:

  • Many HS mimetics lack specificity and can interact with other heparin-binding proteins

  • Well-designed small-molecule inhibitors can achieve higher selectivity, as exemplified by compound 6b (4-Bn-RK-682), which demonstrated selective HPSE inhibition without off-target effects on protein tyrosine phosphatases

What high-throughput screening strategies have been most effective for identifying novel HPSE inhibitors?

Several high-throughput screening (HTS) strategies have proven effective for identifying novel HPSE inhibitors:

• Radiometric assays: Using [³H]-labeled HS immobilized on sepharose beads or streptavidin-coated plates to detect inhibition of HPSE activity through measurement of retained radioactivity .

• Colorimetric assays: Based on specific binding of basic FGF (bFGF) to HS, measuring reduction in bFGF binding as indicated by color development from TMB peroxidase .

• Virtual screening approaches: Computational methods exploiting the available crystal structures of HPSE to identify potential inhibitors through molecular docking and structure-based design .

The effectiveness of these approaches is demonstrated by the discovery of several lead compounds:

• Benzimidazole core lead compound 1 was identified through radiometric HTS

• Isoindole-5-carboxylic acid/benzoxazole compound 7 (IC₅₀: 8 μM) was discovered using colorimetric-based HTS

• Furanylthiazole acetic acid lead compound 12 (IC₅₀: 25 μM) was identified and optimized to yield compounds with IC₅₀ values as low as 300-400 nM

What are the structure-function correlations for HPSE stability mutations that preserve enzymatic activity?

The computational design of stable HPSE variants has revealed important structure-function correlations:

• Mutation distribution patterns: Stability-enhancing mutations are predominantly located on the protein surface, away from the active site and heterodimer interface .

• Structural conservation: Despite introducing 26 mutations in the HPSE P6 variant, the enzyme remained essentially structurally isomorphous to wild-type HPSE, with:

  • No significant changes to the C-α backbone

  • Preserved side chain rotamer sampling

  • Maintained active site geometry

• Dynamic properties: Molecular dynamics simulations confirmed that the engineered variants exhibited dynamics largely identical to wild-type enzyme, indicating no significant changes to the relative stabilities of different conformational substates .

• Functional neutrality: The mutations acquired through phylogenetic analysis reinforced the concept of functional neutrality of many surface residues, as the engineered HPSE variant maintained:

  • Similar catalytic activity

  • Identical inhibition profiles with competitive inhibitors

  • Proper heterodimer formation with the 8 kDa subunit

How can multidimensional data tables be utilized for complex HPSE activity analysis?

Multidimensional data tables offer powerful tools for analyzing complex relationships between multiple variables affecting HPSE activity:

• Application scenarios: This approach is particularly valuable for HPSE research when investigating:

  • Multiple inhibitor concentrations against varying substrate concentrations

  • Effects of pH, temperature, and ionic strength simultaneously

  • Complex interactions between multiple compounds in combination therapy approaches

• Implementation approach:

  • Identify key variables (e.g., inhibitor concentration, pH, temperature)

  • Set up data input with each driver as a row in the table

  • Populate scenario numbers across the top

  • Integrate data input into analysis

  • Construct the data table to generate comprehensive results

• Advantage over multiple simple tables: A single multidimensional data table allows researchers to examine interactions between three or more variables simultaneously, providing more comprehensive insights than could be achieved with multiple two-dimensional tables .

What are the most sensitive detection methods for quantifying low levels of HPSE activity in biological samples?

Detecting low levels of HPSE activity in biological samples requires highly sensitive methodologies:

• Fluorogenic substrate assays: Using synthetic substrates with fluorescent tags that are released upon HPSE cleavage, providing real-time monitoring capabilities with nanomolar sensitivity.

• ELISA-based detection systems: Combining substrate-specific antibodies with enzyme-linked detection for quantification of HPSE-cleaved products in complex biological matrices.

• Mass spectrometry approaches: Analyzing the specific fragmentation patterns of HS oligosaccharides produced by HPSE digestion, allowing for both quantification and structural characterization.

• Enhanced radiometric methods: Modifications of traditional [³H]-labeled HS assays with improved signal amplification and background reduction for detecting extremely low enzyme concentrations.

How should researchers approach the validation of novel HPSE inhibitors across different experimental systems?

A comprehensive validation approach for novel HPSE inhibitors should include:

• In vitro enzyme inhibition characterization:

  • Determination of IC₅₀ values using purified recombinant HPSE

  • Evaluation of inhibition mechanism (competitive, uncompetitive, or non-competitive)

  • Assessment of selectivity against related enzymes and off-target effects

• Cell-based assays:

  • Measurement of HPSE activity inhibition in relevant cell lines

  • Evaluation of effects on cell invasion and migration

  • Assessment of impact on HS-dependent signaling pathways

  • Analysis of compound cytotoxicity and cellular uptake

• Comparative analysis with reference compounds:

  • Benchmark against established HPSE inhibitors like OGT2115

  • Comparison with HS mimetics in terms of potency and selectivity

• Structural characterization of inhibitor binding:

  • X-ray crystallography or molecular modeling to confirm binding mode

  • Surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics and thermodynamics

• Animal model validation:

  • Pharmacokinetic profiling to assess bioavailability

  • Efficacy testing in disease-relevant models (e.g., cancer metastasis, inflammatory conditions)

  • Evaluation of toxicity and safety profile

What emerging technologies might advance the field of HPSE inhibitor development?

Several emerging technologies hold promise for advancing HPSE inhibitor development:

• AI-driven drug design: Machine learning algorithms trained on existing HPSE inhibitor data could identify novel chemical scaffolds and predict activity with greater efficiency than traditional screening approaches.

• Fragment-based drug discovery (FBDD): This approach can identify low molecular weight fragments that bind to different regions of HPSE, which can then be linked or grown to create highly specific inhibitors with improved drug-like properties.

• Cryo-electron microscopy: Advancements in cryo-EM resolution may enable visualization of HPSE in different conformational states, providing insights into dynamics that X-ray crystallography cannot capture.

• Activity-based protein profiling: Development of activity-based probes specific for HPSE could enable direct assessment of inhibitor engagement with active enzyme in complex biological systems.

• Targeted protein degradation: Adapting PROTAC (Proteolysis Targeting Chimera) technology to HPSE could provide an alternative approach to functional inhibition by inducing selective degradation of the enzyme.

How might experimental approaches to studying HPSE activity differ across disease contexts?

Experimental approaches to studying HPSE activity require customization based on disease context:

• Cancer research:

  • Focus on invasion and metastasis assays

  • Analysis of tumor microenvironment interactions

  • Evaluation of combined therapies with existing cancer treatments

  • Assessment of effects on angiogenesis

• Inflammatory diseases:

  • Emphasis on immune cell recruitment and activation

  • Analysis of cytokine release and signaling

  • Evaluation of tissue-specific inflammation models

  • Assessment of HS degradation products as inflammation mediators

• Diabetes and metabolic disorders:

  • Focus on vascular complications and endothelial dysfunction

  • Analysis of basement membrane integrity

  • Evaluation of kidney filtration barrier function

  • Assessment of interaction with glucose metabolism pathways

• Viral infections:

  • Investigation of viral attachment and entry mechanisms

  • Analysis of immune evasion strategies

  • Evaluation of viral replication in presence of HPSE inhibition

  • Assessment of potential as broad-spectrum antiviral approach

Table: Comparative Analysis of Small-Molecule HPSE Inhibitors

*Data compiled from search result