ProFACT

 
SeraFILE™ - A Biomarker and Drug Discovery Engine
SeraFILE™ (patent pending) is a surface-based separations reagent set and associated protocols that addresses the disconnect between proteomics and functional mechanisms. Clients and collaborators now employ it to simultaneously survey the functional characteristics of a targeted protein set for: feature comparisons and discovery, low-abundance enrichment, and for proteomic prospecting.  SeraFILE™ uniquely enables the differentiation of conformational variants and allosteric characterization, critical data for drug development and not otherwise available with other proteomic methods. SeraFILE™ separations and protocols are seamless with existing proteomic assay and detection infrastructure, generating differential sub-proteomes efficiently and in parallel, compartmentalizing the functional characteristics that can define crude soluble protein samples.
The SeraFILE™ surface library is not based on conventional liquid chromatography stationary phases. Whereas conventional LC (green) suffers from a heterogeneous mix of binding energies, and Affinity (red) has exceedingly high binding energy, SeraFILE™ promotes weak, homogeneous binding - ideal for proteomic investigation. Each surface in the library has a singular mixed mode architecture consisting of some combination of ionic, aromatic, aliphatic and polymeric chemistry. Modest pH changes generate unique sub-proteomes with both differential abundance and functional characteristics.
The SeraFILE™ surface library provides:
  • A parallel separations process that is open-ended and industrially productive.
  • Enriched sub-proteomes towards targeted biologically functional conformations.
  • New profiling techniques which generate signatures across a multiplicity of sub-proteomes and interrogations.
  • A means to characterize enzyme regulation and related functional sub-states from disease.
  • Discovery strategies that enrich catalytic activity and directly couple to drug development.
Low Abundance Protein Enrichment
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment.
 
Secondary Interrogation
An immediate and direct hand-off simplifies secondary interrogation. Thus, molecular profiles can be generated from the SeraFILE sub-proteomes, combining function (yellow), structure (blue) and expression and identification (green).
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These measurements collectively provide a molecular profile (or "signature") of the starting sample. Comparisons of a critical number of clinically defined samples, can correlate proteins individually or as a nexus to disease.
Along with comparison analyses, SeraFILE™ participates in three unique discovery avenues especially for biomarker drug development:
  • Rational Proteome Prospecting™, to enrich for low abundance biomarkers of interest,
  • Conformerics™, to compartmentalize conformational variants for small molecule modulation screening assays, and
  • Rational Genome to Proteome Prospecting, for protein biomarker prospecting from gene expression targets.
The discovery of protein biomarkers is severely hindered by the high abundance proteins which are uninteresting and can mask the presence of the putative marker. Due to the increased sensitivity of low molecular weight component analysis, the presence of a protein can oftentimes be determined by measurement of its derivative products, even when no protein marker is evident. SeraFILE™ provides such a rational tiered method for isolating the causative 'needle in the haystack' protein(s). SeraFILE™ can deliver multi-faceted proteomic signatures combining elements of protein abundance as well as functional activities.
These elements can be used to "score" the various pools for enrichment of desirable activity. Each sub-proteome is scored - by bioassay, HPLC, NMR, or mass spectrometry, relative to its total protein content. The high-score fractions are selectively applied to additional tiers of enrichment; each tier producing successively greater levels of purification. Because functional activity is maintained in all pools, Rational Proteome Prospecting™ can be applied to any measurable metabolite or byproduct. Also, through pool recombination, Rational Proteome Prospecting™ techniques can be applied to discover important enzyme regulating factors..
Ubiquitin/Proteasome Pathway (UPP) Characterization.
Background.The 20S proteasome forms the catalytic core of the UPP complex, responsible for proteolytic activity. The 19S regulatory complex associates with either or both ends of the 20S complex. The 19S regulator is involved in the recognition, binding and de-ubiquitinylation of ubiquitinylated proteins tagged for destruction, and stimulates its proteolytic activity.

Results. Western Blot analysis of SeraFILE™ -derived yeast sub-proteomes. Ub(n) regions visualize proteins conjugated to Ubiquitin; Numbers 1-11 correspond to the SeraFILE sub-proteomes; RPT1 regions are indicative of 19S; Bottom Line numbers are measurements of specific proteolytic activity of the 20S particle. Control Proteolytic Activity (without SeraFILE™ treatment) was 82 Units.
Low Abundance Protein Enrichment
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
Conformerics™
Conformational variations are of particular importance in drug target modulation and molecular interaction. Functional Proteomic endeavors could therefore fulfill an urgent pharmacological need, once the regulatory and conformational variability of protein activity can be identified, isolated and measured. ProFACT's SeraFILE™ fulfills this purpose, integrating data-reporting infrasturcture already available, to produce more precisely defined functional proteomic characterizations.
This has immediate value in the discovery, early validation and assay development of biomarkers. Furthermore, as significant conformational events accompany the binding of drugs, functional proteomic profiles offer a direct link to drug development. Consider that a conventional drug development strategy is to lock in the latent (or attenuated) conformation usually through competitive binding to the substrate site; for kinases as an example, the ATP binding pocket is a common target. There are two fundamental deficiences to this strategy: 1) in many enzymes families like kinases, the active domain sites are highly conserved across multiple enzymes and so these drug candidates promiscuously interfere with normal cellular function, and 2) latent states are in much higher relative abundance than their "activated" counterparts, and so drug candidates targeting latent states must have comparatively higher dosages.
SeraFILE can compartmentalize alternate conformers. For drug and biomarker development, it is essential to understand if up-regulated enzyme pathways are due to the overall increase in enzyme abundance, its localization and microenvironments within the cell, allosteric modulation, or changes in its assembly/stability. An important outcome of functional proteomic investigations is to identify the factors that accompany (and potentially cause) higher catalysis when observed in disease samples. This provides a new drug development strategy. Rather than pairing small molecules to the attentuated conformer- the conventional drug discovery route, the ProFACT paradigm would screen small molecules that would pair and modulate alternate conformations. The advantages are that alternate conformations can be more specific to the disease phenotype, and especially when hyperactive conformers can be compartmentalized, lower stoichiometric requirements. These combined advantages would effectively translate to a higher therapeutic index.
Unique and proprietary to ProFACT, these combined deliverables will provide an exceptional new approach to accelerate drugs to market. Rational Proteome Prospecting ™ can be used to isolate or purify important drug targets. Conformerics™ will integrate these targets within drug screening assays.
SeraFILE™-derived enzymatic activity profiles provide a means to detect disease-specific conformers and compartmentalize them for drug screening purposes.
Proteasome chymotryptic activity increased significantly in cancer samples in most sub-fractions, for breast (A) and esophageal cancer (data not shown), compared to normal adjacent tissue (the control). Red arrows indicate altered proteasome activity in a few notable sub-proteomes compared to control extracts (for instance flow-through fractions from matrix 1 and 11; B).
Altered proteasome activity in certain sub-fractions may have clinical relevance because 1) disease-specific inhibitors (i.e. potential drug targets) may be identified in other fractions and 2) activity profiles may function as disease-specific biomarkers.
SeraFILE™ Service Deliverables
SeraFILE™ can be advantageous in routine proteomic investigations using 1DE/2DE/Mass Spec approaches, or through ProFACT's proprietary menu of services. Deliverables are client driven and can include:
  • Differential Sub-proteome pools
  • Molecular Disease Signatures
  • Bioassay Profiles
  • Reproducible Quantitative Abundance Profiles
  • Functional Pathway Characterization
  • Rational Proteome Prospecting™
  • Conformerics™ Bioactive Target/Drug Assays


 
SeraFILE™