Costs associated with populating the drug pipeline are, as always, on the rise. Success demands that pharmaceutical research be linked to the biology of the human system as closely as possible and as soon as possible in the candidate-selection process.
Biomarkers that reflect both the state of the targeted disease as well as the unperturbed system have proven to be informative and more easily manipulated and quantified as combinatorial technologies emerge.
As more companies scramble to fill the void left by pulled-off-the- market products (e.g., Vioxx) and clinical trial failures, rapid, accurate and sensitive identification of metabolites, analysis of new configurations of parent drugs, and isolation of proteins with intact post-translational structures will be among the key requirements of the discovery process.
Optimized Analytical Methods
Determining the configurations of and modifying structures with known clinical utility constitute another effective strategy for generating new leads. Mass spectrometry (MS) and its adjunct methodologies have added growing precision and versatility to molecular research, delivering specific and quantitative results of immediate use to the pharmaceutical researcher.
To keep pace with the productivity requirements of metabolism studies in discovery, optimized analytical methods and efficient data interpretation tools are essential. Thus, pharmaceutical companies have increased their focus on early characterization of metabolites and identification of potential indicators of toxicity in drug discovery.
Scientists at Waters (Milford, MA), and their collaborators in the pharmaceutical industry, have applied Ultra Performance Liquid Chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF) to early metabolism studies.
Increasing throughput and information content for in vitro drug metabolism experiments by using UPLC coupled to Q-TOF/MS has produced an increase in metabolite peak resolution and spectral quality over high-performance liquid chromatography/mass spectrometry (HPLC/
MS), according to the company.
The extra resolution and sensitivity afforded by coupling UPLC with mass spectrometry reduces the risk of missing potentially toxic or reactive metabolites.
Applied Biosystems (Foster City, CA) couples its hybrid triple quadrupole/linear ion trap mass spectrometer, the Q TRAP LC/MS/MS System, into the BIOiTRAQ QT system to enable faster identification, characterization, and quantitation of biomarkers.
Sally Webb, proteomics marketing manager for the Americas at Applied Biosystems, says that this technology is a "hybrid system combining triple quadrupole sensitivity and specificity with ion trap technology, heightening confidence in biomarker identification and validation.
"In addition to biomarker ID and expression analysis with iTRAQ reagents, the system allows multiple reaction monitoring (MRM) workflows that provide specific and sensitive quantitation."
Webb adds that the Q TRAP system's MRM mode has been used successfully to "detect and quantitate biomarkers of interest in hypertension, such as angiotensin II in plasma. Since many drugs involve the inhibition of the conversion of angiotensin I to angiotensin II," this biomarker is of obvious interest to monitor vascular health.
Further applications are in "studying a family of Cytochrome P450 with an eye to predicting or monitoring an individual's reaction to a drug" based on that person's P450 profile.
As there is great genetic variability in one's endowment of these P450's, the company "has built a sensitive MRM assay that can analyze several isoforms responsible for the majority of drug metabolism in one experiment. This is unique in that the assay is looking directly at the proteins rather than indirectly by mRNA or enzyme substrate inhibition," continues Webb.
In effect, this approach constitutes the first protein assay of its kind in its research phase, according to Webb. Post-translational modifications, such as phosphorylation of peptides/proteins, are important in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.
"The Q TRAP system, because of its true hybrid nature, includes the highly specific precursor ion and neutral loss scans that target these modified peptides (triple quadrupole scans)," adds Webb.
"These, combined with the highest sensitivity ion trap MS/MS scans means you can target, detect, and quantitate phosphorylation of peptides/proteins in a single experiment."
Best in Class Therapeutics
Syrrx (San Diego) focuses on "higher validation state" drug targets for which efficacies have been demonstrated in early clinical trials. Often these clinically validated targets have unknown 3-D structures.
Syrrx has a proprietary high throughput structural biology (HTSB) platform that quickly identifies 3-D structures of the protein targets and exploits this information to develop therapeutics.
The Syrrx approach has yielded new kinase structures quickly, and the company has had success in synthesizing novel inhibitors of histone deacetylators involved in oncology, as well as inhibitors of dipeptidyl peptidases for diabetes treatment.
In the analytical step of the platform process, Syrrx uses open-access liquid chromatography/ mass spectrometry (LC/MS) to support protein expression and purification, coupled with capillary LC/tandem mass spectrometry (LC/MS/MS) to characterize protein crystals produced by Nanovolume Crystallization and identify their preferentially expressed protein isoforms.
In addition, the company uses numerous walk-up LC/MS systems that are available to the medicinal chemistry staff to support small molecule analysis and preparative purification.
Mass-directed purification, a technique pioneered by members of Syrrx' analytical team, has proven to be a key technology that spans hit generation, lead series identification, and lead optimization.
Supercritical fluid chromatography/MS (SFC/MS) is another tool used by Syrrx, primarily during the lead optimization process, for separations of chiral enantiomers. SFC is an attractive alternative to HPLC for these enantiomeric separations.
A low viscosity, highly compressible binary solvent system of carbon dioxide and methanol is used, resulting in faster separations as well as reduced solvent usage and disposal costs.
Dan Kassel, Ph.D., senior director of analytical and discovery technologies at Syrrx, indicates that Syrrx has some "interesting chiral separation techniques in development that are both higher throughput and more intelligent."
All of these analytical tools facilitate rapid generation and characterization of high-quality molecules (greater than 95% pure based) that are directly amenable to biological, in vitro ADME profiling and in vivo pharmacokinetic screening.
Rapid discrimination of potential leads is aided by the use of "intelligent" Metabolite Identification (ID) software, developed in collaboration with Applied Biosystems.
Metabolic stability and metabolite ID data are generated in a fully automated and unattended manner, and the software platform is supported on both the API4000 triple quadrupole LC/ MS/MS and QTRAP mass spectrometers.
In addition, Syrrx continues to develop novel, higher throughput analytical methods to support the drug discovery process.
Dr. Kassel, a patent holder in the area of parallel purification methods, claims that Syrrx is "a leader on mass-directed purification techniques," and analytical technologies like these have contributed to shortening of the timeline from hit to clinical candidates selection.
"Our goal is to routinely go from target selection to the clinic in under three years (in contrast to the industry average of five years currently required)," states Dr. Kassel. "For our DPP IV diabetes program, we progressed from the first cloning of the gene to the start of clinical trials in 30 months."