Slow Association Kinetics Research Articles

Design, synthesis and biological evaluation of novel thiosemicarbazones as cruzipain inhibitors

Based on the activity of 23 TSCs on CZ taken from the literature, we have developed a QSAR model for predicting the activity of TSCs. New TSCs were designed and then tested against CZP, resulting in inhibitors with IC50 values in the nanomolar range. The modelling of the corresponding TSC-CZ complexes by molecular docking and QM/QM ONIOM refinement indicates a binding mode compatible with what was expected for active TSCs, according to a geometry-based theoretical model previously developed by our research group. Kinetic experiments on CZP suggest that the new TSCs act by a mechanism that involves the formation of a reversible covalent adduct with slow association and dissociation kinetics. These results demonstrate the strong inhibitory effect of the new TSCs and the benefit of the combined use of QSAR and molecular modelling techniques in the design of new and potent CZ/CZP inhibitors.

Interdomain dynamics in human Replication Protein A regulates kinetics and thermodynamics of its binding to ssDNA.

Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions.

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

Slow Receptor Binding of the Noncytopathic HIV-2UC1 Envs Is Balanced by Long-Lived Activation State and Efficient Fusion Activity

Many HIV strains downregulate the levels of CD4 receptor on the surface of infected cells to prevent superinfection. In contrast, the rare HIV-2UC1 strain is noncytopathic and has no effect on CD4 expression in infected cells but still replicates as efficiently as more cytopathic strains in peripheral blood mononuclear cells (PBMCs). Here, we show that HIV-2UC1 Env interactions with the CD4 receptor exhibit slow association kinetics, whereas the dissociation kinetics is within the range of cytopathic strains. Despite the resulting 10- to 100-fold decrease in binding affinity, HIV-2UC1 Envs exhibit long-lived activation state and efficient fusion activity. These observations suggest that HIV-2UC1 Envs evolved to balance low affinity with an improved and readily triggerable molecular machinery to mediate entry. Resistance to cold exposure, similar to many primary HIV-1 isolates, and to sCD4 neutralization suggests that HIV-2UC1 Envs preferentially sample a closed Env conformation. Our data provide insights into the mechanism of HIV entry.

(S)-5-(2'-Fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, a Serotonin Receptor Modulator, Possesses Anticonvulsant, Prosocial, and Anxiolytic-like Properties in an Fmr1 Knockout Mouse Model of Fragile X Syndrome and Autism Spectrum Disorder.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disabilities and a plethora of neuropsychiatric symptoms. FXS is the leading monogenic cause of autism spectrum disorder (ASD), which is defined clinically by repetitive and/or restrictive patterns of behavior and social communication deficits. Epilepsy and anxiety are also common in FXS and ASD. Serotonergic neurons directly innervate and modulate the activity of neurobiological circuits altered in both disorders, providing a rationale for investigating serotonin receptors (5-HTRs) as targets for FXS and ASD drug discovery. Previously we unveiled an orally active aminotetralin, (S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an Fmr1 knockout mouse model of FXS and ASD, FPT is prophylactic for audiogenic seizures. No FPT-treated mice displayed audiogenic seizures, compared to 73% of vehicle-treated mice. FPT also exhibits anxiolytic-like effects in several assays and increases social interactions in both Fmr1 knockout and wild-type mice. Furthermore, FPT increases c-Fos expression in the basolateral amygdala, which is a preclinical effect produced by anxiolytic medications. Receptor pharmacology assays show that FPT binds competitively and possesses rapid association and dissociation kinetics at 5-HT1ARs and 5-HT7Rs, yet has slow association and rapid dissociation kinetics at 5-HT2CRs. Finally, we reassessed and report FPT's affinity and function at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.

Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158

Arginase-1 is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Arginase-1 is abundantly expressed by tumor-infiltrating myeloid cells that promote tumor immunosuppression, which is relieved by inhibition of Arginase-1. We have characterized the potencies of the Arginase-1 reference inhibitors (2S)-2-amino-6-boronohexanoic acid (ABH) and Nω-hydroxy-nor-L-arginine (nor-NOHA), and studied their pH-dependence and binding kinetics. To gain a better understanding of the structural changes underlying the high pH optimum of Arginase-1 and its pH-dependent inhibition, we determined the crystal structure of the human Arginase-1/ABH complex at pH 7.0 and 9.0. These structures revealed that at increased pH, the manganese cluster assumes a more symmetrical coordination structure, which presumably contributes to its increase in catalytic activity. Furthermore, we show that binding of ABH involves the presence of a sodium ion close to the manganese cluster. We also studied the investigational new drug CB-1158 (INCB001158). This inhibitor has a low-nanomolar potency at pH 7.4 and increases the thermal stability of Arginase-1 more than ABH and nor-NOHA. Moreover, CB-1158 displays slow association and dissociation kinetics at both pH 9.5 and 7.4, as indicated by surface plasmon resonance. The potent character of CB-1158 is presumably due to its increased rigidity compared to ABH as well as the formation of an additional hydrogen-bond network as observed by resolution of the Arginase-1/CB-1158 crystal structure.

Direct observation of individual tubulin dimers binding to growing microtubules

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

Direct Observation of Individual Tubulin Dimers Binding to Growing Microtubules

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

DNA Polymerase Inhibition by High Kinetic Stability of T-HgII-T Base Pairs.

A fluorescent surrogate of thymidine called DMAT was used for the first fluorescence-based study of HgII binding to discrete T-T sites in duplex DNA. The fluorescent properties of DMAT-A base pairs were highly sensitive to wild-type T-HgII-T base pair formation at an adjacent site, allowing for a determination of the precise thermodynamic and kinetic parameters of these metal binding reactions. T-HgII-T complexes exhibited equilibrium dissociation constants of Kd ≈ 8-50 nM. These high-affinity binding interactions are characterized by very slow association and dissociation kinetics (kon ≈ 104- 105 M-1s-1, koff ≈ 10-4 - 10-3s-1), revealing exceptionally high kinetic stabilities of T-HgII-T base pairs (half-lives = 0.3-1.3 h). Duplex DNA containing DMAT and no T-T mismatch exhibited nonspecific HgII binding affinities of Kd ≈ 2.0 μM. The high kinetic stabilities of T-HgII-T resulted in the inhibition of dynamic processes such as DNA strand invasion and strand displacement during enzymatic DNA synthesis, which led to premature chain termination. These results demonstrated that T-HgII-T base pairs are kinetically distinct from T-A base pairs and therefore are likely to disrupt DNA metabolism in vivo.

Significance of Steric Bulk in DNA Threading Intercalation Revelaed through Force-Dependent Kinetics

DNA threading intercalation is a DNA binding mode, whereby a molecule must thread a bulky group through the DNA duplex to allow a planar central section to stack with the DNA base pairs. Molecules that bind to DNA via threading intercalation display incredibly slow association and dissociation kinetics. This is a result of the large duplex deformations required for the ligand to thread and unthread the DNA. While this binding mode has been investigated extensively, the specific molecular characteristics that affect the binding to DNA are still poorly understood. This study utilizes single molecule stretching with optical tweezers to elucidate the effects that the bulk and geometry of the threading moieties have on DNA binding. Single molecule optical tweezers experiments are a powerful tool in analyzing threading intercalation because they allow for direct quantification of both the equilibrium behavior and kinetics of DNA-ligand binding. The equilibrium binding profile of the binuclear ruthenium complex ΔΔ-[μ-bidppz(bipy)4Ru2]4+ (ΔΔ-B) was quantified in this study, revealing a dissociation constant on the same order of magnitude as its previously studied, bulkier sister molecule ΔΔ-[μ-bidppz(phen)4Ru2]4+ (ΔΔ-P). ΔΔ-B shows faster on rates compared to ΔΔ-P at low force, while both on rates converge to comparable values at high force. The off rates of ΔΔ-B show no force dependence, whereas the off rates of ΔΔ-P are strongly facilitated by force. These results suggest that in the absence of force that ΔΔ-B threads DNA faster and unthreads slower, making ΔΔ-B a more ideal DNA binding anticancer drug.

Long Residence Times Revealed by Aurora A Kinase‐Targeting Fluorescent Probes Derived from Inhibitors MLN8237 and VX‐689

We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well-known inhibitors MLN8237 and VX-689 (MK-5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 10(5)-10(7) M(-1) s(-1), koff 10(-3)-10(-4) s(-1); residence time 500-3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on-rate and increase in the off-rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX-689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.

A New Quantitative Bead Aggregation Assay for Determining the Association Rates of Protein-Protein Interactions

Developing new quantitative methods for studying the interactions of cell surface proteins is imperative to advance understanding of the molecular determinants of cell adhesion. Our interest in the dimerization kinetics of cadherin, a calcium-dependent cell-cell adhesion molecule, has motivated us to develop a quantitative bead aggregation assay to study the effects of calcium concentration on the kinetics of association. Although bead aggregation has been used for some time to study protein-protein interactions, the data are usually only interpreted qualitatively. Here we establish a quantitative method for studying the aggregation of beads that are coated with neural-cadherin. Data indicated exponential growth in aggregate size as a function of time. Time constants of association ranged from 18 to 3 minutes over a 100 fold increase in calcium concentration. We used this method to study the slow association kinetics of a mutant cadherin (NCAD1/R14E). These studies yielded a time constant of 28 minutes, which indicates that the association rate of mutant is slower than wild type (9 minutes). Experimental factors were controlled to ensure precision in the data including protein binding to beads, incubation time in the presence of calcium, mixing of the beads prior to placing on the slide and the residence time on the slide before data were acquired. This method is the first report of experimental determination of the kinetics of the association of proteins tethered to a surface, and is broadly applicable to study the kinetics of protein-protein interactions.

Transcription factor binding kinetics constrain noise suppression via negative feedback

Negative autoregulation, where a transcription factor regulates its own expression by preventing transcription, is commonly used to suppress fluctuations in gene expression. Recent single molecule in vivo imaging has shown that it takes significant time for a transcription factor molecule to bind its chromosomal binding site. Given the slow association kinetics, transcription factor mediated feedback cannot at the same time be fast and strong. Here we show that with a limited association rate follows an optimal transcription factor binding strength where noise is maximally suppressed. At the optimal binding strength the binding site is free a fixed fraction of the time independent of the transcription factor concentration. One consequence is that high-copy number transcription factors should bind weakly to their operators, which is observed for transcription factors in Escherichia coli. The results demonstrate that a binding site's strength may be uncorrelated to its functional importance.

Kinetic Signatures of Metals in the Presence of Suwannee River Fulvic Acid

This work provides new information on the dissociation kinetics of metal-fulvic acid (FA) complexes. Diffusive gradients in thin-film (DGT) devices deployed in solutions containing metals and 30 mg L(-1) Suwannee River FA at pH 5 and 7, at two different metal-to-ligand ratios, were used to estimate an apparent diffusive boundary layer (ADBL) thickness at the gel-solution interface. The discrepancy between the ADBL thickness measured for metals that are known to dissociate from complexes quickly (e.g., Cd) and that of other trace metals was exploited to calculate the rate of complex dissociation. When the ADBL thickness is plotted for a suite of metals, a "kinetic signature" is created. There was a clear kinetic signature at pH 7, with substantial kinetic limitation for Cu, Pb, and Ni and none for Cd, Co, and Mn (i.e., Cu-, Pb-, and Ni-FA complexes dissociated more slowly). At pH 5, the kinetic signature was less distinct, due in part to slow association kinetics of Mn, and possibly Cd and Co, with the resin. The good sensitivity of the method to small changes in dissociation kinetics was able to show that the dissociation of most metal-FA complexes is sufficiently fast to not limit the DGT measurement.

Revealing the Affinity of Individual and Combined Flig Domains for Flim in the Bacterial Flagellar Motor Switch Complex by CG-MALS

Complex interactions between flagellar motor proteins modulate the rotational direction of the bacterial flagella. In particular, the middle and C-terminal domains of FliG (FliGM and FliGC, respectively) bind two different sites on the binding partner FliM, as part of the flagellar motor switch. We extend previous NMR studies of the interactions between FliG domains with FliM via composition gradient multi-angle static light scattering (CG-MALS) to confirm specific binding, quantify affinity and identify the stoichiometry of each complex. Though the equilibrium association constants between the individual FliGC domains and FliGM differ by >25x, both complexes appear to reach equilibrium on time scales faster than that of the CG-MALS measurement (∼5 sec). By contrast, binding of the dual-domain protein FliGMC to FliM exhibited unusually slow association kinetics with reaction time constants on the order of 30-60 minutes. CG-MALS revealed complex self-and hetero-association stoichiometries that could not be identified or quantified by means of NMR. Combined with structural studies, these data could provide insight to the mechanism of flagellar motor switching.

Meso-tetraphenyl porphyrin derivatives: The effect of structural modifications on binding to DMPC liposomes and albumin

Three series of glycoconjugated and hydroxylated derivatives of 5,10,15,20- meso-tetraphenyl porphyrin (TPP) were studied in order to evaluate the effect of a porphyrin structure on its binding to dimyristoylphosphatidylcholine (DMPC) liposomes and to human serum albumin (HSA). The studied derivatives have been developed as potent photosensitizers for photodynamic therapy (PDT) of cancers. Steady state and time resolved fluorescence emission spectroscopy, Stern–Volmer quenching and fluorescence anisotropy were used for this evaluation. The lipophilicity of the compounds has been deduced from their retention time in reverse phase liquid chromatography. The results demonstrated that the more polar glycoconjugated compounds presented limited aggregation in aqueous media and very rapid binding kinetics to DMPC liposomes and HSA. Derivatives having intermediate or high hydrophobicity showed extensive auto-association in aqueous media and as a consequence slow association kinetics. The strength of porphyrin binding to DMPC liposomes also depended on their lipophilicity and was lower for the polar glycoconjugated analogues. The highest affinity for liposomes was observed for hydroxylated derivatives with intermediate lipophilicity. In contrast, the highest binding constant for albumin was observed for a polar tetra-glycoconjugated analogue. The depth of penetration into the phospholipid bilayer did not appear to be directly related to the global hydrophobicity of the compounds, but depended more on the number of apolar, non-substituted phenyl groups grafted to a tetrapyrrolic macrocycle. Furthermore, liposome–albumin competition studies revealed that the porphyrins were always mainly partitioned into the phospholipid bilayer.

Structural, thermodynamic, and kinetic effects of a phosphomimetic mutation in dynein light chain LC8.

Dynein light chain LC8 is a small, dimeric, very highly conserved globular protein first identified as an integral part of the dynein and myosin molecular motors but now recognized as a dimerization hub with wider significance. Phosphorylation at Ser88 is thought to be involved in regulating LC8 in the apoptotic pathway. The phosphomimetic Ser88Glu mutation weakens dimerization of LC8 and thus its overall ligand-binding affinity, because only the dimer binds ligands. The 1.9 A resolution crystal structure of dimeric LC8(S88E) bound to a fragment of the ligand Swallow (Swa) presented here shows that the tertiary structure is identical to that of wild-type LC8/Swa, with Glu88 well accommodated sterically at the dimer interface. NMR longitudinal magnetization exchange spectroscopy reveals remarkably slow association kinetics (k(on) approximately 1 s(-1) mM(-1)) in the monomer-dimer equilibrium of both wild-type LC8 and LC8(S88E), possibly due to the strand-swapped architecture of the dimer. The Ser88Glu mutation raises the dimer dissociation constant (K(D)) through a combination of a higher k(off) and lower k(on). Using a minimal model of titration linked to dimerization, we dissect the thermodynamics of dimerization of wild-type LC8 and LC8(S88E) in their various protonation states. When both Glu88 residues are protonated, the LC8(S88E) dimer is nearly as stable as the wild-type dimer, but deprotonation of one Glu88 residue raises K(D) by a factor of 400. We infer that phosphorylation of one subunit of wild-type LC8 raises K(D) by at least as much to prevent dimerization of LC8 at physiological concentrations. Some LC8 binding partners may bind tightly enough to promote dimerization even when one subunit is phosphorylated; thus linkage between phosphorylation and dimerization provides a mechanism for differential regulation of binding of LC8 to its diverse partners.

Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.

Enzyme Fragment Complementation Binding Assay for p38α Mitogen-Activated Protein Kinase to Study the Binding Kinetics of Enzyme Inhibitors

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.