How To Figure Out Binding From K App

Binding Kinetics

Besides the binding kinetics and receptor distribution, peripheral clearance, regional cerebral blood flow (CBF), and transport across the blood–brain barrier will influence the profile of the time–activity curve.

From: Encyclopedia of Neuroscience , 2009

Drug Discovery Technologies

Y. Fang , in Comprehensive Medicinal Chemistry III, 2017

2.17.5.3 SPR to Characterize the Binding Kinetics of Lead-Like Compounds

Binding kinetics is concerned with the rate constants of ligand association ( k _on) and dissociation (k _off); and the ratio of the two defines the equilibrium dissociation constant (K _d = k _off/k _on). Compared to binding affinity, the kinetics, in particular receptor residence time, of a drug has found to be better correlated with its clinical efficacy, safety, duration of action, tolerability, indication, and therapeutic differentiation. ¹⁵ ^, ¹⁶ For instance, drugs with slower off-rates give longer duration of action, more likely leading to a once-a-day dosing. For instance, the long lasting anti-bronchiodilatation drug tiotropium dissociates 10 times slower from its primary target, muscarinic M₃ receptor, than from M₁ and M₂ receptors, resulting in an improved selectivity for this drug over other mixed M₁/M₂/M₃ antagonists. ¹¹⁴ Tiotropium exhibits similar binding affinity at both M₂ and M₃ receptors; the M₃ receptor in airway tissues is the primary target for its efficacy, while the M₂ receptor in body is responsible for its cardiovascular side effects.

The drugs such as tiotropium that work well because of kinetic differences were not designed that way. Thus, understanding kinetics earlier prospectively would be of great value, since it gives access to more diverse chemical space, more scope for intellectual property, and better defined biology. SPR is the standard technique now routinely used in laboratories for characterizing the affinity and kinetics of drugs binding to target proteins in vitro. SPR is applicable for a wide range of protein classes, particularly soluble proteins including enzymes, kinases, and proteases. However, applying SPR to GPCRs has been slow. ¹¹⁵ Recently, the use of thermostablized or tagged wild-type GPCRs has made it possible to use SPR to characterize ligand–GPCR interactions including adenosine A_2A receptor ¹¹⁶ and β₁-AR, ¹¹⁷ both of which contain a number of point mutations to improve thermostability and are further stabilized in a specific conformation in complex with a specific ligand.

Biolayer interferometry (BLI) is an alternative for profiling binding kinetics, mostly for antibodies. Instead of microfluidics used in SPR, BLI dips optical microfibres directly into the wells of standard plates for kinetic measurements with relatively higher throughput. ¹¹⁸ Given that there is plentiful evidence showing that compounds with the same affinity but different on- and off-rates can have a very different biological activity profile, there is a strong demand for high-throughput kinetic profiling techniques.

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Microtubules, in vitro

Bhabatarak Bhattacharyya , ... Dulal Panda , in Methods in Cell Biology, 2010

2 Determination of Association Rate Constant Using Quenching of Intrinsic Tubulin Fluorescence

Binding kinetics of all colchicinoids, whether they fluoresce in the presence of tubulin or not, can be measured using ligand-induced quenching of intrinsic tubulin fluorescence. Quenching of fluorescence caused by the binding of all colchicinoids is due to resonance energy transfer from the excited tryptophan of tubulin to the bound colchicinoid. Resonance energy transfer requires an overlap between the fluorescence emission spectrum of the donor (here tubulin) and the absorption spectrum of acceptor (colchicinoids). The association of the colchicinoids with tubulin has been measured under pseudo-first-order condition using intrinsic fluorescence quenching. Quenching of intrinsic protein fluorescence was measured at 336 nm with an excitation wavelength of 280 nm (Chakraborty et al., 2004). Under these experimental conditions, no aggregation of tubulin was detected by the size-exclusion high-performance liquid chromatography (HPLC) column. Temperature was controlled with circulating water bath accurate to ±0.5°C. All fluorescent measurements were carried out in a 0.5-cm path length quartz cuvette, and fluorescence values were corrected for the inner filter effect according to Lakowicz (1999),

$F_{corr} = F_{obs} \times antilog (\frac{A_{280} + A_{336}}{2})$

where F _obs and F _corr are the observed and the corrected fluorescence values and A ₂₈₀ and A ₃₃₆ are the absorbance values at the excitation and emission wavelengths, respectively. The value ln (Q _max – Q_t ) was plotted against time (Fig. 6A and B), Q _max is the maximum amount of quenched fluorescence, and Q_t is the quenched fluorescence at time t. The biphasic plot was analyzed according to the method of Lambeir and Engelborghs (1981),

$Q_{\max} - Q_{t} = A \cdot e^{- α t} + B \cdot e^{- β t}$

where A and B are amplitudes and α and β are observed rate constants of the fast and slow phases, respectively. The amplitude of the slow phase (B) was low relative to that of the fast phase (A) and the slow phase was not analyzed further. The apparent association rate constants (k _on) were calculated as k _on = α/c, where α is the slope of the plot of ln (Q _max – Q_t ) versus time t and c is the concentration of ligand.

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Volume I

Thomas J. Gardella , ... John T. PottsJr., in Endocrinology: Adult and Pediatric (Seventh Edition), 2016

Conformationally Selective Parathyroid Hormone Ligands and Prolonged Signaling

Kinetic binding and signaling assays performed using various PTH and PTHrP ligand analogues provide data to suggest that the PTHR-1 can indeed adopt different protein conformations that display differential affinities for structurally diverse ligands, and can thereby mediate different types of signaling responses. ^373-376 In particular, these studies show that certain PTH analogues can form complexes with the PTHR-1 that maintain a high affinity state, even upon the addition of GTPγS, a reagent that promotes the dissociation of G proteins from the receptor and thus typically is used to shift a GPCR into a low-affinity state. These PTH ligand analogues are thought to bind to a novel PTHR-1 conformation, called R⁰, that can maintain high affinity even independently of G protein coupling. In contrast, other ligands, such as the shorter-length M-PTH(1-14) analogues, form complexes with the PTHR-1 that rapidly dissociate upon addition of GTPγS. ³⁷³ Accordingly, these PTH ligands are thought to bind primarily to a G protein–coupled conformation called RG.

The biological consequences of such ligand-directed conformational selectivity at the PTHR-1 are not evidenced by a change in signaling pathway type (e.g., from the Gαs/cAMP/PKA pathway toward a non–Gαs-mediated pathway), but rather by the duration of the cAMP response that is induced by the different ligands. Thus, R⁰-selective ligands are seen to induce prolonged cAMP responses in PTHR-1-expressing cells, whereas RG-selective PTH ligands induce more transient cAMP responses that diminish soon after initial ligand exposure. While the complete mechanisms underlying the prolonged signaling responses observed for the R⁰-selective ligands are far from clear, a potential explanation is that they arise from the capacity of the ligand to remain bound to the receptor through multiple and repeated rounds of coupling to Gαs. In any event, the effects are robust and observable not only in cell-culture systems, but also in animals, in which R⁰-selective ligands have been shown to induce increases in blood calcium levels and decreases in blood phosphate levels that persist for at least several hours longer than those observed for PTH(1-34), even when the R⁰ analogue is injected at a dose severalfold lower than the dose used for PTH(1-34). ^374,376

One long-acting analogue of particular interest, called LA-PTH, is a hybrid peptide composed of the M-PTH(1-14) sequence joined to the PTHrP(15-36) sequence. ³⁷⁶ LA-PTH binds to the R⁰ PTHR-1 conformation with severalfold higher affinity than does PTH(1-34), and while the cAMP potency measured for LA-PTH is the same as that measured for PTH(1-34), consistent with their equivalent RG-binding affinities, the cAMP response induced by LA-PTH persists for many hours longer than that induced by PTH(1-34). When injected into mice, LA-PTH induces elevations of serum calcium that can last for 24 hours or longer, whereas PTH(1-34) injection at the same dose results in responses that last only a few hours (Fig. 56-10). Importantly, pharmacokinetic studies have indicated that the prolonged responses to such R⁰-selective PTH analogues are not due to prolonged durations of the peptides in the circulation, ⁸⁸ and so they are more likely to be due to a persistent binding of the ligands to the PTHR-1 in bone and kidney target cells. Because of their prolonged actions in vivo, the class of such R⁰-selective PTH analogues would appear to hold promise as a potential new line of therapy for patients with hypoparathyroidism. ¹³⁵

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Gradients and Tissue Patterning

Anqi Huang , Timothy E. Saunders , in Current Topics in Developmental Biology, 2020

Without binding kinetics (or other processes inhibiting diffusion), FCS and FRAP return similar estimates of diffusion coefficients ( Sigaut et al., 2014). However, in most biological systems this is rarely the case. It has been argued that since FRAP reports on longer timescales, it is more biologically relevant (Grimm et al., 2010). However, the short time kinetics of the morphogen are important, for both dispersal and signal interpretation (Sigaut et al., 2014). Measurements of the lifetime and decay length of the Bcd gradient find values around 30 min (Durrieu et al., 2018) and 85 μm (Little et al., 2011; Liu et al., 2013) respectively. This gives an effective diffusion constant of around 3 μm² s^{− 1}. Both FCS (D _Bcd ~ 5–10 μm² s^{− 1}) and FRAP (D _Bcd ~ 0.3–1 μm² s^{− 1}) provide useful information but neither, by itself, is able to definitively describe Bcd dynamics as D is an amalgamation of numerous processes, including diffusion, nuclear trapping, binding, and potential active transport through the cytoskeleton.

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Globins and Other Nitric Oxide-Reactive Proteins, Part B

Stefania Abbruzzetti , ... Cristiano Viappiani , in Methods in Enzymology, 2008

1 INTRODUCTION

Ligand binding kinetics following nanosecond laser photolysis reflects the ligand migration inside the protein matrix and the reactivity toward molecules diffusing from the bulk solution. Although O ₂ is the physiological ligand of myoglobin (Mb) and hemoglobin (Hb), it is often difficult to avoid irreversible oxidation or side reactions while studying these very reactive species in vitro. In model studies, carbon monoxide (CO) is often preferred to oxygen as a ligand, as it does not further react chemically with the heme after binding to the Fe atom. CO generally binds even more strongly than O₂ to the heme and its complexes are extremely stable in the long term. The Fe-CO bond is photolabile, and the use of short (nanosecond) light pulses allows a large population of reactive deoxy states to be generated transiently and the rebinding of CO over time to be monitored spectroscopically. The time span of this process is generally extended over several orders of magnitude in time. Immediately after the end of the laser pulse, the ligand undergoes competitive reactions in which it can either move through the protein matrix and temporarily dock to hydrophobic pockets or react with the heme to form the carboxy adduct. The presence of kinetic traps results in nonexponential relaxations, and analysis of the time course in geminate recombination can yield useful mechanistic information on the ligand migration pathways. The amplitude of this kinetic phase is extremely variable, depending on how easy it is for the photodissociated ligand to escape to the bulk solution. To overcome the problem of small geminate yields, proteins can be encapsulated in silica gels. Under these conditions, geminate recombination is often enhanced, particularly for monomeric proteins, and the sensitivity to migration processes is accordingly increased. For multimeric proteins, such as the tetrameric human hemoglobin A, substantial enhancement of the geminate phase can be observed when encapsulated in silica gels under increased viscosity, for instance, in the presence of glycerol.

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Molecular Characterization of Autophagic Responses, Part A

F. Fassy , ... B. Pasquier , in Methods in Enzymology, 2017

4.1 Assay Principle and Protocol

The binding kinetics of compounds were evaluated by surface plasmon resonance (SPR) on the N-terminal GST tag full-length protein. In addition, a specific study was performed to compare the full-length and the truncated 282–879 protein used for crystallization, to ascertain that both forms had similar molecular interactions with the compounds.

The experiments were achieved on ProteOn XPR36 (Biorad). SPR sensors measure refractive index changes occurring at the sensor surface during a course of biomolecular interaction. SPR involves immobilization of one of the binding partner (usually named ligand, i.e., VPS34 protein) on the sensor surface, while the other binding partner (named analyte, i.e., small molecule) remains in solution. The interaction of an analyte with a ligand increases the refractive index at the surface and is directly proportional to the amount of bound analyte. This change in refractive index is expressed in terms of response units (RU). The interaction between an analyte and the ligand is monitored in real time and is represented by a plot, or sensorgram, of RU vs time. The ProteOn XPR36 has a unique approach to multiplexing; this system generates a 6 × 6 interaction array for the simultaneous analysis of up to six ligands with up to six analytes.

Each new sensor chips were preconditioned by three subsequent washes using 50 mM NaOH, 10 mM HCl, and 0.1% SDS. The protein immobilization was performed on GLM sensor chip using standard amine coupling procedure in PBS, 0.05% Tween 20, immobilization running buffer (Biorad). Amine coupling requires preparation of the protein in a low-salt buffer at least 0.5 pH unit below the protein isoelectric point. After an 8-min activation of carboxylic groups by a 1:1 mixture of 1 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.25 M N-hydroxysuccinimide sulfo to give reactive succinimide esters, the protein was injected at a concentration of 16 μg/mL (full length) or 20 μg/mL (truncated) in 10 mM MES buffer at pH 6 in the presence of an inhibitor of intermediate affinity to protect the exposed lysines of the active site. During this step, the esters reacted spontaneously with the amino or other nucleophilic groups to link the ligand to the surface of the chip. Free surface activated carboxyl groups remaining after covalent modification were quenched with 1 M ethanolamine pH 8.5 during 5 min. Typical immobilization levels ranged from 8500 to 11,500 RU and from 6500 to 7300 RU for the truncated and full-length protein, respectively. A nonderivatized surface (without ligand) was used as reference for bulk refractive index correction.

All interactions of small molecules (analytes) were studied at 25°C. The compounds were tested at six concentrations (typically from 10 × K _D to 0.3 × K _D). The running buffer was made of 50 mM HEPES-NaOH, pH 7.1, 5 mM MgCl₂, 150 mM NaCl, 2 mM TCEP, and it was supplemented with 2% DMSO. Analyte injections were performed at a flow rate of 100 μL/min for a 1-min association phase, followed by a 5-min dissociation phase. DMSO has a high refractive index, e.g., a 0.1% variation in DMSO concentration between the sample and the running buffer translates to 100 RU of SPR signal. It was therefore necessary to add a DMSO calibration curve for bulk effect and "excluded volume" corrections. Typical DMSO calibration curve ranged from 1.5% to 3.5% DMSO. A buffer blank control was also included for baseline drift correction.

The determination of the kinetic parameters was performed according to the recommendations of the constructor (http://www.bioradiations.com/guide-to-spr-data-analysis-on-the-proteon-xpr36-system/). First, the reference surface and the buffer blank responses were subtracted from all the interaction data collected over the different reaction surfaces and the "excluded volume" corrections was applied. From the corrected sensorgram data, the rate constants were determined by the ProteOn software using a Langmuir 1:1 binding model, in which one analyte molecule (A) interacts reversibly with one ligand molecule (B), with the following assumptions: pseudo-first-order kinetics, equivalence and independence of binding reactions, no limitation by mass transport. The equilibrium dissociation constant (K _D) is the ratio between two rate constants, K _D = k _d/k _a, with k _a, the association rate constant, and k _d, the dissociation rate constant. The kinetic analysis of the association and dissociation phases of the sensorgram was based on Eqs. (4) and (5), respectively.

(4) $R_{t} = \frac{R_{max} [A]}{K_{D} + [A]} [1 - e^{- (k_{a} [A] + k_{d}) t}]$

(5) $R_{t} = R_{0} e^{- k_{d} t},$

where R_t , R _max, and R ₀ are the responses as time t, at the maximum of the association phase for a saturating concentration of analyte, and at the beginning of the dissociation phase, respectively.

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Cell Biophysics

L. S.-L. Cheung , ... K. Konstantopoulos , in Comprehensive Biophysics, 2012

Glossary

2-D binding kinetics: The binding kinetics the binding kinetics between membrane-bound receptors and ligands anchored to apposing cell surfaces, whose motions are restricted to a 2-D plane.
Bell model: A model proposed by Bell for describing receptor–ligand bond dissociation, wherein the bond dissociation rate increases exponentially with the applied force.
Catch bond: receptor–ligand bonds whose lifetimes increase upon the application of a low external force.
Dembo model: A model proposed by Dembo for describing receptor–ligand bond dissociation, wherein the bond lifetime may decrease or increase or remain the same upon application of an external force.
Micropippette aspiration assay: A technique to measure the two-dimensional (2-D) kinetics of receptor–ligand interactions by characterizing the adhesion frequency as a function of contact duration between two apposing cells.
Molecular force probe: A device that utilizes a flexible cantilever, which deflects in response to forces generated between the tip and the sample surface when brought together, for measuring the kinetic and micromechanical properties of receptor–ligand bonds at the single molecule level.
O-glycans: The modification of serine or threonine residues on proteins by addition of a GalNAc residue, which results in an O-linked oligosaccharide or O-glycan.
PSGL-1: The P-selectin glycoprotein ligand-1 (PSGL-1; CD162) is a membrane glycoprotein that is expressed on virtually all blood leukocytes, human hematopoietic progenitor cells, and to a much lesser extent on blood platelets.
Selectin: A Ca²⁺-dependent transmembrane glycoprotein present on the surface of circulating leukocytes, activated platelets, and endothelial cells at sites of inflammation/infection.
Shear threshold phenomena: A counterintuitive phenomenon in which the extent of cell binding to selectins first increases and then decreases while monotonically increasing the wall shear stress.
Shear-controlled association rate: An association rate model wherein the shear-induced slipping motion of a cell relative to a wall may enhance the encounter rate between membrane-bound receptors and immobilized ligands.
sLe^x: The tetrasaccharide sialyl Lewis x {sLe^x; NeuAc α2,3 Gal β1,4 (Fuc α1,3) GlcNAc-R} is a terminal component of glycans attached to glycoproteins and glycolipids on most circulating leukocytes, and on some endothelial cells and tumor cells.
Slip bond: receptor–ligand bonds whose lifetimes decrease with the application of an external force.
Two-pathway dissociation model: A model wherein the unbinding of the selectin–ligand complex from a single bound state can be either along the catch pathway over a low-energy barrier or the slip pathway along a high-energy barrier.

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Drug Discovery Technologies

G.A. Holdgate , in Comprehensive Medicinal Chemistry III, 2017

2.07.2.25 Future Trends for Kinetic Characterization

Currently, designing a particular binding kinetics profile into a compound remains challenging but clearly remains a strategic goal for the medicinal chemist, as kinetics provides an opportunity to optimize compounds to gain a therapeutic advantage. Modifying binding kinetics can lead to improved therapeutic index and improve coverage via changes in PK/PD relationships.

Modifying binding kinetics will ultimately require changes to the energy levels of either the transition state or the protein–ligand complex, and understanding how to achieve these will be key. The increased measurement of binding kinetics, together with both structural information and computational approaches such as molecular modeling and molecular dynamics simulations, will help to provide a better understanding of both protein dynamics and the factors influencing binding kinetics.

Currently, we often think about lead optimization from an equilibrium perspective, since the interactions that are introduced relate to the two ends of an equilibrium, and we understand little of what happens in between. We may consider the modification of a compound structure and the result on residence times in two simplistic ways: Firstly, the structural change leads to a conformational change in the protein, leading to altered binding kinetics; secondly, the structural change changes the balance of interactions to favor the slowly dissociating ones. In this simplistic view, it would appear that the former would be very target-specific and understanding the effect on protein conformation might be challenging to accomplish in order to have impact in medicinal chemistry projects in a timescale that would be useful. The second approach seems more tractable, and we would hope that there may be some simple guidelines that could be established to promote rational design.

And even if this cannot be done directly, the strategy for optimizing long residence times can focus on optimizing affinity or by exploring additional binding interactions that are associated with larger kinetic barriers.

As our understanding of the implication of binding kinetics grows, the opportunity to impact projects will improve as we begin to implement kinetic optimization in order to decrease the number of iterations to move from lead to candidate drug or from first-in-class to best-in-class molecule.

Several recent articles have reviewed the current status of the use of binding kinetics in drug discovery for improved drug effects, ⁴⁴ focusing on optimizing on rates, ⁴⁵ and for GPCR kinetic optimization. ⁴⁶

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Kinetics of Drug-Target Binding: A Guide for Drug Discovery

Sam R.J. Hoare , in Reference Module in Biomedical Sciences, 2021

5.4 Case study of mis-estimation of affinity owing to lack of equilibration—CRF₁ receptor antagonist development

A practical example of how binding kinetics impacts SAR assays in drug discovery is provided by a recent case-study. This study described the consequences of lack of equilibration, how it was discovered, and how properly accommodating binding kinetics resulted in the development of a clinically-effective molecule ( Hoare et al., 2020a). The target was the corticotropin releasing factor type 1 receptor (CRF₁ receptor), a GPCR. Antagonists for this receptor were being developed by numerous groups for the treatment of neuropsychiatric and endocrine disorders, based on the role of CRF as a primary behavioral and endocrine regulator of the stress response (Gilligan et al., 2000; Grigoriadis, 2005; Kehne and De Lombaert, 2002). In the case study, an effective molecule had been identified (NBI 30775) but was discontinued for toxicological reasons. An SAR campaign was in progress to identify new active molecules. The efficacy of NBI 30775 was evident in preclinical and clinical endpoint and biomarker measurements (Chen et al., 2004; Gutman et al., 2003; Heinrichs et al., 2002; Held et al., 2004; Zobel et al., 2000). Fig. 15 shows the activity of the compound in the animal model used to test in vivo efficacy, inhibition of adrenocorticotropin (ACTH) release in rats. (CRF released from the hypothalamus acts on pituitary corticotropes to stimulate ACTH production and release into the circulation.) NBI 30775 produced a large and sustained inhibition of ACTH release in the model (Fleck et al., 2012; Schwandt et al., 2016); 2 h after dosing, ACTH was reduced by 91% at the highest dose tested (30 mg/kg) and inhibition was sustained for at least 6 h (Fig. 15). The dose response was quantified using the AUC of the time course from 0 to 4 h. From this, the % ACTH inhibition was calculated, then this was plotted against the AUC of the compound concentration in the plasma (Fig. 16).

New compounds were being synthesized and tested. These compounds were tested in an in vitro competition binding assay to measure affinity for the CRF₁ receptor. The conditions were typical for a binding assay used in drug discovery—the assay was incubated for 90 min at room temperature (see (Fleck et al., 2012) for details). Five new compounds were selected for in vivo testing in the rat ACTH assay based on the K _i in the binding assay. The measured in vitro K _i for all compounds was similar to that of NBI 30775 (10 nM, see Fig. 16 for new compound K _i values). The new compounds had improved pharmacokinetic properties compared with NBI 30775, for example higher plasma exposures (Hoare et al., 2020a). However, when tested in vivo, the compounds were much less active than NBI 30775 despite the higher plasma concentrations in the animals (Fig. 16). Numerous factors were tested to see if they could account for the lack of effect, including protein binding and species selectivity, but none of the factors could explain it. The disconnect between in vitro and in vivo activity of the compounds was a considerable problem for the project because there was no way to predict the in vivo activity of the molecules using in vitro assays.

At this time the target binding properties of the antagonists were being evaluated using radiolabeled analogues of the compounds. ³H-NBI 30775 had been synthesized, and as part of the standard characterization of radioligand binding a dissociation assay was conducted. This assay revealed that dissociation of ³H-NBI 30775 was remarkably slow (Fleck et al., 2012). At room temperature, the temperature used to quantify affinity of the compounds, the dissociation t½ was more than 5 h. In fact, only 10% of the radioligand had dissociated by the 5 h time point (Fleck et al., 2012). From this value, an extrapolated dissociation t½ of 33 h can be calculated. This is an unusually slow dissociation event for a GPCR ligand (compare with values in Table 1). The implication of this result was that the assay used to quantify NBI 30775 affinity was very far from equilibrium, the incubation time of 90 min being far less than the calculated dissociation t½ of 33 h. This meant that the affinity of NBI 30775 was likely underestimated substantially, which could potentially explain the in vivo results. If the true affinity of NBI 30775 was much higher, this could explain why it was more potent in vivo than the new compounds.

In order to test this hypothesis it was necessary to develop methods to accurately quantify the affinity, methods that could be accommodated within the daily workflow of a drug discovery project. This is very challenging when the residence time is so long. Fortunately, the problem was made more tractable by increasing the assay temperature. At 37 °C, dissociation was faster, the dissociation t½ of ³H-NBI 30775 reduced to 3.2 h (Fleck et al., 2012). However, this was still too long to be accommodated by a standard equilibrium binding assay—to closely approach equilibrium and so accurately measure affinity an incubation time of three times the dissociation t½ is needed, which translated to an incubation time of 9.6 h. To solve this problem, the affinity was measured kinetically. As described in Section 2.3, affinity is related to the binding kinetics by the equation K _d/i = k _off/k _on. A method was developed to measure k _off and k _on of the new compounds using the competition kinetics approach (Motulsky and Mahan, 1984; Aranyi, 1980) (as described in Section 6.2.3). Unlabeled test compound was competed against ³H-NBI 30775 at 37 °C at multiple time points, and data fit to an equation to determine k _off and k _on of the unlabeled compound (Fleck et al., 2012). These k _off and k _on values were then used to calculate the K _i, which was termed "Kinetic K _i."

These experiments demonstrated that the affinity of NBI 30775 was much higher than originally estimated under the non-equilibrium conditions of the binding assay. The kinetic K _i was 0.76 nM, compared with 10 nM under the nonequilibrium conditions (Hoare et al., 2020a; Fleck et al., 2012). They also demonstrated that the affinity of the new compounds was much lower than that of NBI 30775, ranging from 18 to 140 nM (Hoare et al., 2020a). This dramatic difference between the initial non-equilibrium K _i results and the true K _i from the kinetic assay is illustrated in Fig. 17A . This result provided a simple explanation for why the new compounds were less active in vivo – they simply bound with a lower affinity. (The experiments also demonstrated a lower affinity of some compounds at 37 °C compared with room temperature, potentially a consequence of the markedly accelerated dissociation at 37 °C.) We extended this comparison to literature standards (Fig. 17B) (Fleck et al., 2012). Eleven compounds were tested, compounds advanced to clinical or late preclinical testing that had been assumed to bind with similar affinity using the conventional in vitro binding assay. The measured affinity under non-equilibrium conditions in a side-by-side comparison varied by ninefold (Fig. 17B). However, the kinetic assay revealed the true affinity range was much broader, at 500-fold (Fig. 17B) (Fleck et al., 2012). Thus the kinetic affinity assay revealed previously unappreciated differences between compounds that are used extensively to examine CRF₁ receptor physiology and pharmacology.

Now that the problems of the in vitro/in vivo potency disconnect and of measuring affinity accurately had been solved, attention was turned to identifying compounds which were as active as NBI 30775 in the in vivo model. For this purpose, the kinetic competition assay was used to quantify k _off, k _on and the kinetic K _i of the compounds. This provided a convenient in vitro assay for triaging compounds to test in vivo. Compounds were identified that bound with kinetic K _i in the range of that for NBI 30775 and that dissociated slowly from the receptor. These compounds were tested in the in vivo rat ACTH model and numerous molecules were identified that were similarly potent to NBI 30775 for suppressing ACTH. One of these compounds, verucerfont (NBI 77860) (Tellew et al., 2010), was advanced to clinical testing. This compound, with a kinetic K _i of 15 nM and dissociation t½ of 1 h (Hoare et al., 2020a), reduced ACTH in healthy human volunteers (Schwandt et al., 2016), and reduced ACTH and downstream hormones in patients with an endocrine disorder, congenital adrenal hyperplasia (Turcu et al., 2016).

This case study demonstrates how knowledge of binding kinetics can impact drug discovery. A problem—inability to predict in vivo efficacy—was solved by discovering that compounds dissociated slowly from the target, indicating the original assay was underestimating affinity owing to lack of equilibration. Incorporating kinetics into the drug discovery cascade by measuring the binding rates and the kinetic K _i enabled good prediction of in vivo efficacy. This resulted in the identification of highly-active new molecules, one of which demonstrated efficacy in a human disease population.

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Immunomagnetic Particle-Based Techniques

K.S. Cudjoe , in Encyclopedia of Food Microbiology (Second Edition), 2014

Incubation During IMS

During incubation, intermittent mixing enhances binding kinetics, resulting in the isolation and separation of higher levels of target organisms than when static incubation is used. Continuous mixing during incubation gives even better binding kinetics, resulting in increased recovery of target organisms. The fear of cross-contamination from the tube's cap has been expressed as a basis for static incubation without closing tubes. Static incubation could be used for those IMS protocols that involve less than 3 min incubation, but the beads must be mixed thoroughly with the sample before static incubation commences, otherwise they will sediment. A major source of contamination, however, is the use of contaminated wash buffer, improper handling of pipettes and lack of good laboratory practice. Care should be taken to distinguish between wash buffers meant for first and second washes and from those meant for reconstitution of particles after the final wash. Main batch wash buffers should be aliquoted into smaller volumes and used appropriately. Care must be taken to avoid the development of aerosols while aspirating sample supernatants or wash buffer supernatants or when adding wash buffers. When opening Eppendorf tubes, the index finger should be placed over the cap and the thumb should be used gently to lift it upward, or an opening device for Eppendorf tubes should be used.

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