Quantitation of physiological and biochemical barriers to siRNA liver delivery via lipid nanoparticle platform.

Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50's ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.

Integrating the Impact of Lipophilicity on Potency and Pharmacokinetic Parameters Enables the Use of Diverse Chemical Space during Small Molecule Drug Optimization.

Lipophilicity has a dominant effect on many parameters that determine unbound drug exposure as well as drug potency. Despite this, analysis of a large body of drug data indicates lipophilicity has no consistent directional impact on dose. This can be rationalized based on the interplay of the effects of lipophilicity on individual parameter values in pharmacokinetic equations. We believe this undermines the effectiveness of strategies that target specific ranges for drug parameters for which lipophilicity plays such a dominant role. As a result, our research organization no longer leverages the common approach of screening for low intrinsic clearance in vitro to target high unbound exposure in vivo. Instead, we advocate for approaches less biased to lipophilicity through optimization of key parameter ratios controlling dose. We believe this improves efficiency in drug discovery by enabling exploration of broad physicochemical space.

Remote electronic control in the regioselective reduction of succinimides: a practical, scalable synthesis of Ep4 antagonist MF-310.

A practical large-scale chromatography-free synthesis of EP4 antagonist MF-310, a potential new treatment for chronic inflammation, is presented. The synthetic route provided MF-310 as its sodium salt in 10 steps and 17% overall yield from commercially available pyridine dicarboxylate 7. The key features of this sequence include a unique regioselective reduction of succinimide 2 controlled by the electronic properties of a remote pyridine ring, preparation of cyclopropane carboxylic acid 3 via a Corey-Chaykovsky cyclopropanation, and a short synthesis of sulfonamide 5.

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was > or = 2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies.

INTRODUCTION: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [35S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide ([35S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. METHODS: Functional potencies of unlabeled compounds were characterized by [14C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. RESULTS: ACPPB is a potent (Kd=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [35S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [35S]ACPPB in rat brain tissues following iv administration of this radioligand. CONCLUSIONS: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission tomography tracers for GlyT1.

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B1 receptor antagonist.

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.

LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.

In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Discovery of N-{N-[(3-cyanophenyl)sulfonyl]-4(R)-cyclobutylamino-(L)-prolyl}-4-[(3',5'-dichloroisonicotinoyl) amino]-(L)-phenylalanine (MK-0668), an extremely potent and orally active antagonist of very late antigen-4.

Extremely potent very late antigen-4 (VLA-4) antagonists with picomolar, whole blood activity and slow dissociation rates were discovered by incorporating an amino substituent on the proline fragment of the initial lead structure. This level of potency against the unactivated form of VLA-4 was shown to be sufficient to overcome the poor pharmacokinetic profiles typical of this class of VLA-4 antagonists, and sustained activity as measured by receptor occupancy was achieved in preclinical species after oral dosing.

A general and efficient 2-amination of pyridines and quinolines.

Pyridine N-oxides were converted to 2-aminopyridines in a one-pot fashion using Ts2O-t-BuNH2 followed by in situ deprotection with TFA. The amination proceeded in high yields, excellent 2-/4-selectivity, and with good functional group compatibility. 2-Amino (iso)quinolines were also obtained in the same manner. Combined with the simple oxidation of pyridines to pyridine N-oxides, this method provides a general and efficient way for amination of 2-unsubstituted pyridines.

Synthesis of the first sulfur-35-labeled hERG radioligand.

The synthesis of the first high specific activity S-35-labeled hERG radioligand, [(35)S]MK-0499, for use in HTS assays of drug candidates for hERG interaction is described. The radioligand is prepared by [(35)S]sulfonylation of a high diastereomeric excess (de) aniline precursor prepared from unlabeled MK-0499.

Identification and characterization of isomeric intermediates in a catalyst formation reaction by means of speciation analysis using HPLC-ICPMS and HPLC-ESI-MS.

Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process.

Induction of CYP1A in the beagle dog by an inhibitor of kinase insert domain-containing receptor: differential effects in vitro and in vivo on mRNA and functional activity.

Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (approximately 11.3 microM . h), relative to day 1 (28.2 microM . h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.

Mechanism-based inhibition of human liver microsomal cytochrome P450 1A2 by zileuton, a 5-lipoxygenase inhibitor.

Zileuton, a 5-lipoxygenase inhibitor, was evaluated as an inhibitor of cytochrome P450 activity in human liver microsomes. In the absence of preincubation, the racemate was found to be a weak inhibitor (IC50 > 100 microM) of phenacetin O-deethylation (POD) (CYP1A2), paclitaxel 6alpha-hydroxylation (CYP2C8), diclofenac 4'-hydroxylation (CYP2C9), (S)-mephenytoin 4'-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4), chlorzoxazone 6-hydroxylation (CYP2E1), and bupropion hydroxylation (CYP2B6). When preincubated with NADPH-fortified human liver microsomes in the absence of substrate, zileuton (racemate) was shown to inhibit POD. The effect was NADPH-, time-, and concentration-dependent, and was characterized by a kinact (maximal rate of enzyme inactivation) and apparent KI(inhibitor concentration that supports half the maximal rate of inactivation) of 0.035 min(-1) and 117 microM, respectively (kinact/KIratio of 0.0003 min-1 microM(-1)). Preincubation-dependent inhibition of POD activity was also observed with the individual (S)-(-)- and (R)-(+)-enantiomers of zileuton [(S)-(-)-zileuton; kinact, 0.037 min(-1), KI, 98.2 microM, kinact/KIratio, 0.0004 min(-1) microM(-1); (R)-(+)-zileuton; kinact, 0.012 min(-1), KI, 66.6 microM, kinact/KIratio, 0.0002 min(-1) microM(-1)]. In addition, the inhibition of CYP1A2 was not reversed in the presence of reduced glutathione, catalase, and superoxide dismutase and was refractory to dialysis. Therefore, zileuton was characterized as a mechanism-based inhibitor of human liver microsomal CYP1A2. Mechanism-based inhibition of CYP1A2 may explain why zileuton decreases the oral clearance of antipyrine, propranolol, (R)-warfarin, and theophylline, at doses that have a minimal effect on the pharmacokinetics of (S)-warfarin, phenytoin, and terfenadine.

Metabolism and pharmacokinetics of a dipeptidyl peptidase IV inhibitor in rats, dogs, and monkeys with selective carbamoyl glucuronidation of the primary amine in dogs.

The pharmacokinetics and metabolism of the l-threo isoleucine thiazolidide dipeptidyl peptidase IV inhibitor, di-[2S,3S]-2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate (ILT-threo) and its allo stereoisomer (ILT-allo) were evaluated in rats, dogs, and monkeys. Both compounds were well absorbed (>80%) in all species, and most of the dose (>60%) was recovered in urine. Metabolites identified in all species included a sulfoxide (M1), a sulfone (M2), and a carbamoyl glucuronide (M3). For both compounds, parent drug had moderate systemic clearance in rats and dogs ( approximately 20-35 ml/min/kg in both species) and lower clearance in monkeys ( approximately 6-9 ml/min/kg). In rats, M1 was present in systemic circulation in concentrations similar to that of parent drug, whereas in dogs and monkeys, exposures to M1 were higher than for parent drug. In dogs, exposures to the sulfoxide metabolite were approximately 2 to 3 times higher after administration of ILT-allo than after administration of ILT-threo. Carbamoyl glucuronidation was an important biotransformation pathway in dogs. Circulating levels of M3 were significant in the dog, and present only in trace levels in rats and monkeys. M3 could be produced in in vitro systems in a NaHCO3 buffer under a CO2-saturated atmosphere and in the presence of UDP-glucuronic acid and alamethicin.

Evaluation of ubiquinone concentration and mitochondrial function relative to cerivastatin-induced skeletal myopathy in rats.

As a class, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors can potentially cause skeletal myopathy. One statin, cerivastatin, has recently been withdrawn from the market due to an unacceptably high incidence of rhabdomyolysis. The mechanism underlying statin-induced myopathy is unknown. This paper sought to investigate the relationship among statin-induced myopathy, mitochondrial function, and muscle ubiquinone levels. Rats were administered cerivastatin at 0.1, 0.5, and 1.0 (mg/kg)/day or dose vehicle (controls) by oral gavage for 15 days. Samples of type I-predominant skeletal muscle (soleus) and type II-predominant skeletal muscle [quadriceps and extensor digitorum longus (EDL)], and blood were collected on study days 5, 10, and 15 for morphological evaluation, clinical chemistry, mitochondrial function tests, and analysis of ubiquinone levels. No histological changes were observed in any of the animals on study days 5 or 10, but on study day 15, mid- and high-dose animals had necrosis and inflammation in type II skeletal muscle. Elevated creatine kinase (CK) levels in blood (a clinical marker of myopathy) correlated with the histopathological diagnosis of myopathy. Ultrastructural characterization of skeletal muscle revealed disruption of the sarcomere and altered mitochondria only in myofibers with degeneration, while adjacent myofibers were unaffected and had normal mitochondria. Thus, mitochondrial effects appeared not to precede myofiber degeneration. Mean coenzyme Q9 (CoQ9) levels in all dose groups were slightly decreased relative to controls in type II skeletal muscle, although the difference was not significantly different in most cases. Mitochondrial function in skeletal muscle was not affected by the changes in ubiquinone levels. The ubiquinone levels in high-dose-treated animals exhibiting myopathy were not significantly different from low-dose animals with no observable toxic effects. Furthermore, ubiquinone levels did not correlate with circulating CK levels in treated animals. The results of this study suggest that neither mitochondrial injury, nor a decrease in muscle ubiquinone levels, is the primary cause of skeletal myopathy in cerivastatin-dosed rats.

Development of an efficient and selective radioligand for bradykinin B1 receptor occupancy studies.

We have developed an efficient and selective radioligand, the [35S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B1 receptor.

Identification of metabolites of a substance P (neurokinin 1 receptor) antagonist in rat hepatocytes and rat plasma.

[3R,5R,6S]-3-(2-cyclopropyloxy-5-trifluoromethoxyphenyl)-6-phenyl-1-oxa-7-azaspiro[4.5]decane is a substance P (Neurokinin 1 receptor) antagonist. Substance P antagonists are proven in concept to have excellent potential for the treatment of major depression, and they allow superior and sustained protection from acute and delayed chemotherapy-induced emesis. The metabolism of this compound was investigated in rat hepatocytes, and circulating rat plasma metabolites were identified following oral and intravenous dosing. The turnover in rat hepatocytes within 4 h was about 30%, and the major metabolites were identified as two nitrones and a lactam associated with the piperidine ring. Although these metabolites were also observed in rat plasma, the major circulating metabolite was a keto acid following oxidative de-amination of the piperidine ring. Liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance were used to confirm the structure of the latter metabolite. A mechanism leading to the formation of the keto acid metabolite has been suggested, and most intermediates were observed in rat plasma.

Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1.

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.

A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.

An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.