Commitment to Covalency: Kinetics of Irreversible Inhibitors with a Regenerable Streptavidin Sensor on the Pioneer FE System

Transcription

1 APPLCATON NOTE 23 Commitment to Covalency: inetics of rreversible nhibitors with a Regenerable Streptavidin Sensor the Pieer FE System Aar Martin, Applicatis Manager, Pall ForteBio LLC and Phillip Schwartz, PhD, Senior Scientist, Taeda, CA NTRODUCTON The principal role of assay groups in drug discovery is to provide reliable methods, analysis, and data for cfident decisi-maing about series progressi. Particular assays are chosen to differentiate between affinity, specificity, cellular acti, and most importantly mechanism of acti. ncreasingly, drug discovery faces challenges from harder-to-drug targets alg with regulatory pressures to increase safety, efficacy, and to improve drug ADME (absorpti, distributi, metabolism, excreti) properties. The respses to these challenges come through the creative use of both emergent and standard technologies, investing in new discovery approaches (e.g. fragments), and increasing explorati of drugs that act through targeted covalent modificati. Approximately 3% of the therapeutic enzyme inhibitors the maret functi through covalent modificati of the target. Ccerns of specificity, metabolite reactivity, immunogenicity, clearance, redox activity, etc., often blunt interest in pursuing covalents as a primary discovery strategy. Ctributing to the higher barrier to portfolio entry is a lesser familiarity with targeted covalent inhibitors and an underdeveloped biochemical toolit for characterizing and describing the mechanism of acti for this class of compounds. Factors ctributing to the potency of irreversible inhibiti are poorly understood. An incorrect assumpti often made is that covalent inhibitors d t require the intrinsic potency needed for n-covalent inhibitors since the reacti will mae the binding permanent. Rather, the efficiency of the irreversible reacti relies the specificity and potency of the initial encounter complex (binding). f the encounter complex does not form lg enough for covalent bd formati to occur then chemistry can t assist. Therefore, the necessary wor of maing a potent, ncovalent binding scaffold is still critical. Similarly, understanding the covalent reacti in the ctext of the full drug and protein system is imperative since many factors can influence the rate and extent of reacti. The simple two-step covalent inhibitor model (Equati 1) taes the same mathematical form as traditial Michaelis-Menten inetics, and as such deriving an algebraic soluti is n-trivial for highly optimized inhibitors. Figure 1: Rate equatis for irreversible inhibiti, where E is enzyme, is inhibitor, E is encounter complex, and E- is adduct or covalent complex. and off are rate cstants of associati and dissociati, respectively. is the reacti rate cstant of covalent ivati. The equilibrium approximati assumes that equilibrati of diffusi ctrolled reactants to the final ccentratis of substrate and product is instantaneous. Similarly, the quasisteady state approximati assumes that intermediate complex forms much faster than the cversi to product. Neither of these assumptis are valid as the rate of covalent bd formati overtaes the rate of inhibitor dissociati; this is the goal of any successful covalent drug program. A more comprehensive model must be utilized and experimental methods to derive the cstants developed. However, biochemical assays cannot unambiguously reveal the balance between binding and reactivity, further complicating structure-activity relatiship (SAR) decisimaing and additial informati is necessary to resolve the ctributi of each compent. Combining the results from multiple orthogal techniques has proved particularly powerful in early discovery. n recent years, biophysical methods have made greater ctributis both ale and in ccert with other modalities. n this applicati note we highlight the wor of Dr. Phillip Schwartz, Ph.D. of Taeda California, 1

2 COMMTMENT TO COVALENCY: NETCS OF RREVERSBLE NHBTORS ON THE PONEER FE SYSTEM who champis covalent approaches in drug discovery. Through his recent papers and presentatis, such as the 216 Drug Discovery Cference discussed here, Dr. Schwartz combines the results from biochemical and biophysical experiments to provide a set of intuitive metrics for chemists to trac as part of their SAR around covalent modifiers. Analyzing data obtained from the Pall ForteBio Pieer FE platform s implementati of Surface Plasm Resance (SPR) resolves inherent ambiguities in the biochemical data while providing additial parameters such as the direct observati of and a metric for covalent reacti efficiency (C c ). SPR has enjoyed a growing role in drug discovery and provides direct observati of binding associati and dissociati, in additi to quantitative inetic parameters and a wealth of qualitative data discernable from curve shape. t can be used for fragment screening, mechanistic studies through competiti assay formats, -going program SAR, and assessing hits identified by orthogal technologies especially with activity assays for their direct binding compents and stoichiometry. Characterizing covalent binders with SPR is unusual given that the target protein is typically immobilized to the sensor chip and reused for different compounds and compound ccentratis. This recycling of the surface has generally targeted the technology at reversible systems and SPR labs often refrain from testing covalent modifiers over ccerns of surface fouling. One method that avoids this ccern is reversible affinity capture, which captures target and allows periodic replacement with fresh protein. However, affinity capture methods such as antibody or His-tag capture are often associated with drifting baselines which are detrimental for analyzing the inetics of a covalent inhibitor. Capture of biotinylated target by immobilized streptavidin gives a desirable stable baseline, but regenerati of the surface for periodic replacement of target has proven inefficient as the biotin streptavidin interacti is very high affinity and difficult to reverse. Specialized streptavidin reagents have been used with limited success but the reagents are costly and may not provide the necessary capture density to observe small molecule interactis. These issues can be overcome by capturing target-streptavidin complex to a biotin sensor chip and regenerating with a harsh reagent which removes the protein ctent and leaves the surface ready for capture of fresh target protein. Experimental methods to stably capture target and analyze inetics for irreversible inhibitors will be described. This applicati note delineates Dr. Schwartz s method for combining a reversible streptavidin-biotin capture method with the realtime direct observati of binding/reacti using the Pieer FE system with biochemical progress data to extract a full descripti of the inhibitor s protein associati, dissociati, reactivity and their relative balance. This increased granularity should be invaluable to anye pursuing covalent inhibitor SAR. Reversible nhibiti i i How fast the compound binds to the enzyme off Potency + Figure 2: Equatis derived by Dr. Schwartz to describe the inetics of ivati by covalent binders. i and describe inhibitor potency for reversible and irreversible inhibiti, respectively. is the rate of covalent reacti to form the adduct, E-. By this model e sees that both affinity ( i ) for the target, as well as highly efficient chemistry ( ) are required to get efficient irreversible inhibiti. The C c term introduced is the commitment to covalency representing the probability that the encounter complex proceeds to form the adduct. The product of C c and the associati rate cstant,, describes the overall biochemical potency ( / ). THEORY AND RESULTS Dr. Schwartz s analytical method details ctributis to an irreversible inhibitor s biochemical potency ( / ), a parameter analogous to biochemical i, D or C 5, that accounts for the extended covalent mechanism. mportantly, his model (Figure 2) allows for a compound to bind and partiti to covalent bd formati with high efficiency. Traditial biochemical analysis relies models that do not differentiate this type of highly efficient behavior, hence requiring abstracti beyd Michaelis-Mentenlie inetics. ntroduced is the intuitive parameter commitment to covalency or C c which relates to the efficiency of adduct (E-) formati and its balance with dissociati. t is both the efficiency of adduct formati and the rate of protein/inhibitor associati that defines biochemical potency. Dr. Schwartz then demstrates that this C c parameter can be used as a diagnostic for the rate limiting step in the reacti. n Figure 3, he describes situatis where: Case 1) neither binding nor reacti are efficient, Case 2) binding is more efficient, but the reacti is still not and compounds dissociate far more frequently than they react, Case 3) reacti is faster but binding is inefficient and Case 4) binding is potent and reacti is efficient. Unfortunately, the biochemical rate data ( obs ) ale loo the same for Cases 1 and 4 or 2 and 3. There is still an ambiguity remaining, but SPR can be used to resolve it. The SPR respse curves are evaluated for the distorti to the pure binding/dissociati curves usually obtained for reversible inhibitors tested Pieer technology and shown in Figure 4. n the top off How liely the E complex proceeds toward covalency rreversible nhibiti off + + off C c 2

3 Applicati Note 23 off Case 1: off >> Unbalanced off Case 2: off >> Unbalanced i i.8 Cc <<.1.5 Cc < obs obs off off Case 3: off Near Balance Case 4: >> off Balanced.1 < Cc <.6 Cc > obs.2 obs Figure 3: Examples of different Commitment to Covalency (C c ) cases and the observed results in enzyme assay ( obs ) data. Simulatis show that initial rate assays cannot easily distinguish C c ctributis to covalent inhibitors. 3

4 COMMTMENT TO COVALENCY: NETCS OF RREVERSBLE NHBTORS ON THE PONEER FE SYSTEM Cc.1 off 1*.5 4 obs.4 Occupancy Cc << obs.3.2 Occupancy Cc.5 off < Cc < obs Occupancy 6 4 Cc.9 off.1*.2.1 Cc > Figure 4: SPR can unmas C c ctributis. Enzyme assay simulati (left column) and SPR simulati (right column) of covalent compounds with differing C c properties. Unreacted compound in these examples is shown to dissociate rapidly at the end of injecti (1 sec, top row) leaving reacted compound irreversibly bound. Compounds with higher C c properties (middle and bottom rows) have less dissociati and more reacted compound. From the SPR respses e can unambiguously determine biochemical potency, rate of inhibitor associati and the efficiency of the chemical reacti in the encounter complex. 4

5 Applicati Note 23 row the reacti is slow relative to the dissociati (C c <<.1). At the end of the injecti unreacted compound rapidly dissociates leaving behind ly the small fracti that did react. f the off-rate and chemical rate ( ) are similar some dissociati is observed but a larger fracti is retained (middle row,.1 < C c <.6). n the ideal case (Figure 4, bottom row, C c >.6) the entire fracti that binds also reacts and no dissociati is observed. From a P/PD perspective, a fully committed covalent inhibitor can have significant impact dosage versus an inhibitor that must bind and release many times before target ivati occurs. The theory is put to practice using an improved affinity capture method which captures biotin-tagged target to reversibly immobilized streptavidin. The method taes advantage of the Pieer FE system s robust fluidics by regenerating target-streptavidin complex with a harsh reagent. The benefits of this target capture method are: mproved target activity No requirement for specialized streptavidin reagents A reproducible capture yield for many test cycles A biotin-tagged inase target was pre-incubated with streptavidin and captured per the Desthiobiotin Sensor Capture Method outlined below. Binding data were fit with a form of the two-state inetic model where, off, and are determined at multiple analyte ccentratis. C c is also determined by the previously described relatiship of off and. Three published covalent inhibitors were bound to target using this method and significantly different C c values were observed for each. For reference, an endpoint competiti assay described before was used to independently determine /. The ability for both assays to determine / provides an internal csistency chec while the SPR assay further distinguishes C c from. 1 EXPERMENTAL METHODS Desthiobiotin Sensor Capture Method 1 nstall a Pieer COOHV sensor (PS17AFB) and briefly cditi with two injectis each of 1 mm HCl, 5 mm NaOH, and.1% SDS. 2 Couple Desthiobiotin by injecting activati soluti (.2 M EDC +.5 M NHS) for 25 min at 1 µl/min, then injecting 2 mg/ml Amine-PEG 4 -Desthiobiotin (Thermo Fisher Scientific Cat. # or other) in 5 mm HEPES ph 7.4 for 3 min at 5 µl/min, and deactivating by injecting 1M Ethanolamine HCl ph 8.5 for 5 min. 3 Pre-incubate mobiotinylated target protein with streptavidin in a 1:1 or 2:1 molar ratio to form target-streptavidin cjugate. 4 Capture target-streptavidin cjugate by injecting 25 µg/ml sample for 2 1 min at a flow rate of 1 µl/min, to desired capture density. Capture density may be improved by preparing the cjugate in a preccentrati buffer approximately 1 ph unit below the cjugate isoelectric point (p). For best baseline stability, the capture injecti should stop before it reaches full saturati. The surface may require cditiing for Respse (RU) C c.2 C c.6 C c 1 Respse (RU) Respse (RU) Published nhibitors C c / (M -1 s -1 ) SPR Biochemical Assay / (M -1 s -1 ) inase nhibitor 3 4.E E+4 2.1E+5 inase nhibitor 2 1.E E+5 7.3E+5 inase nhibitor 1 4.1E E+5 8.8E+5 Figure 5: SPR data from the Pieer FE system showing three covalent inhibitors binding target with different C c values. Signal after referencing shows stable baselines, suitable for measuring these potent inhibitors and differentiating binding from covalent reacti. Potency parameters are compared between inhibitors and assay methods to show the differences in reacti efficiency. 5

6 COMMTMENT TO COVALENCY: NETCS OF RREVERSBLE NHBTORS ON PONEER FE cycle-to-cycle capture reproducibility, so include 4 6 captureregenerati cycles to establish a reproducible capture signal prior to testing analytes. 5 nject irreversible analyte sample using either a standard injecti or a OneStep njecti. For standard injectis, use a 3 6 sample ccentrati series and inject each sample for 3 6 min at a flow rate between µl/min. For OneStep njectis, inject three sample ccentratis using the 1% sample loop volume setting at a flow rate between µl/min. nclude a 5 15 min dissociati in each injecti cycle. 6 Regenerate the target-streptavidin cjugate by injecting 1 mg/ml iminobiotin dissolved in a 1:1 mixture of 2 mm NaOH with acetitrile for 2 min. 7 Repeat Steps 3 6 for all analyte and reference (buffer) samples. The Desthiobiotin Sensor method was discovered by the team at Taeda and this method enables an optial step-wise capture of streptavidin followed by biotinylated target. This method gives the desired result of stable capture of protein targets, is compatible with many buffer compents and is fully regenerable. Data were referenced using reference channel respse, and buffer blan signal and inetics were fit with the two-state irreversible inetic model (rate equati shown in Figure 1). C c was calculated as shown in Figure 2. both worlds strategy epitomizes the benefits gained from combining orthogal approaches. The parameter set described ( and C c ) is the reasable minimal set of SAR terms that provide a comprehensive descripti of the inhibiti mechanism. The Michaelis-Menten-lie formulati of inhibitor acti threatens to get unwieldy when the steady-state approximatis are removed. The approach described here simplifies the additial terms into the C c term, maing the interpretati more broadly accessible. C c may be to covalent inhibitors what net rate cstants have been to mechanistic enzymology since the last century. Looing forward, it is exciting to imagine the use of the refined gradient injecti mode offered by the Pieer instruments (OneStep njectis) to provide even more precise estimates of the associati-phase informati, and underlying off-rate where labeling is incomplete or there is intrinsic reversibility. OneStep njectis provide for more inetic curvature in the binding associati, and in the irreversible case the inflecti point of the associati may provide additial meaningful internal csistency checs of the reacti data. Additially, new target capture methods create opportunity for the analysis of covalent inhibitors against more targets and target classes. udos to Dr. Schwartz and the team at Taeda for advancing the state of the art for this ind of drug discovery. CONCLUSON The presented wor reveals a useful extensi of the mechanism of covalent binding drugs and provides a practical framewor for significant differentiati between molecules. This proposed method represents an elegant blend of biochemical and biophysical methodologies and utilizes SPR technology in an innovative way to directly acquire the necessary data (, off, ) to resolve the ambiguity that the obs data ale cannot recover. This best of Acnowledgment The authors would lie to acnowledge the help of Dr. John Quinn, formerly of Taeda, in the co-development of the Desthiobiotin sensor regenerati method and SPR methodology. REFERENCES 1 Miyahisa, T Sameshima, MS Hix, Rapid determinati of the Specificity Cstant of rreversible nhibitors ( / ) by Means of an Endpoint Competiti Assay, Angew Chem nt Ed Engl, 54(47): , 215. Pall ForteBio Fremt Boulevard Fremt, CA t: 888.OCTET-75 or Pall ForteBio Europe 5 Harbourgate Business Par Southampt Road Portsmouth, PO6 4BQ, U t: +44-() Pall ForteBio Analytics (Shanghai) Co., Ltd. No. 88 Shang e Road Zhangjiang Hi-tech Par Shanghai, China 2121 t: , Pall Corporati. Pall,, ForteBio, Pieer, OneStep, and NeXtStep are trademars of Pall Corporati. indicates a trademar registered in the USA and indicates a comm law trademar. AN Rev B