A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditions.

Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related SVI-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

Small molecule cytokine mimetics.

A number of reports describe small peptides, and even bona fide small organic molecules, that activate homodimeric cytokine receptors and show cytokine-like activity in vitro and in vivo. These cases can be examined in light of the mechanistic and thermodynamic principles that govern cytokine-receptor activation.

Application of linear free energy relationships to the serpin-proteinase inhibition mechanism.

Linear free energy relationships can be used to link the changes in rate constant for a reaction to changes in the equilibrium caused by alterations in structure. While they have most often been used in the analysis of chemical reactions, they have also been employed to resolve questions in enzymology and protein folding. Here we analyze the reaction of a serpin with a panel of six serine proteinases, and observe that a linear free energy relationship exists between the true second-order rate constant for reaction, k(inh), and the inhibition constant, K(I), indicating that formation of the covalent serpin-enzyme complex may be reversible.

Mapping of IFN-beta epitopes important for receptor binding and biologic activation: comparison of results achieved using antibody-based methods and alanine substitution mutagenesis.

The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.

ELISA methods for the analysis of antibody responses induced in multiple sclerosis patients treated with recombinant interferon-beta.

Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recombinant interferon-beta (IFN-beta), those receiving BETASERON (IFN-beta-1b; E. coli expressed, non-glycosylated, des-Met-1, Cys17Ser recombinant IFN-beta) have a higher incidence of IFN-beta specific antibodies compared to those receiving AVONEX (IFN-beta-1a; mammalian cell-expressed, natural sequence, glycosylated recombinant IFN-beta). The current study reports the development and characterization of ELISAs that detect distinct components of the anti-IFN-beta response in patients' sera, and therefore can potentially be used to characterize the composition of the anti-IFN-beta antibody response. ELISAs were developed using a constant detecting reagent but a variety of IFN-beta-derived test antigens (e.g., native IFN-beta, biotinylated IFN-beta, IFN-beta peptides) and capture methods. Assays were characterized using serum samples from a small number of patients treated with recombinant IFN-beta (either BETASERON or AVONEX). Assays in which IFN-beta was captured via a specific mAb, or in which biotinylated IFN-beta was captured via streptavidin, detected serum antibodies that recognize IFN-beta in its native structural state. In contrast, assays in which IFN-beta was coated directly onto the assay plates detected antibodies that recognize forms of IFN-beta possessing a folded structure distinct from the native structure. Certain epitopes present on native IFN-beta were not represented in these assays in which the test antigen was directly coated on plastic. Antibodies specific for linear epitopes could be detected using linear peptides as test antigens; the locations of these epitopes were mapped by reference to the X-ray crystal structure of IFN-beta-1a. Together, these data show that the mode of antigen presentation employed in IFN-beta ELISAs determines which antibody specificities are detected, and can affect whether or not a given serum sample is identified as positive for anti-IFN-beta antibodies. As a consequence, screening samples in a single ELISA format presenting IFN-beta in a non-native form may lead to underestimation of the incidence of IFN-beta treated MS patients that have generated antibodies specific to the native, active form of the drug.

Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases.

We report the use of structure-based drug design to create a selective erbB-1 (a.k.a. epidermal growth factor receptor) and erbB-2 (a.k.a. neu/her2 growth factor receptor) tyrosine kinase inhibitor. Using the X-ray crystal structure of the ternary complex of the cAMP-dependent Ser/Thr kinase together with a sequence alignment of the catalytic domains of a representative set of Ser/Thr and Tyr protein kinases, we have examined the nucleotide binding site for potential positions to attach an irreversible inhibitor. This information, combined with homology modeling of the erbB-1 and erbB-2 tyrosine kinase catalytic domains, has led to the identification of Cys797 of erbB1 and Cys805 of erbB2, which are structurally equivalent to Glu127 in the cAMP dependant Ser/Thr kinase as potential target residues. The X-ray structure of the cAMP Ser/Thr kinase shows Glu127 to be involved in a hydrogen-bonding interaction with the 2'-OH of the ribose portion of ATP. Using molecular modeling, it was predicted that the Cys side chains in erbB-1 and erbB-2 performed an analogous role, and it was postulated that the replacement of the 2'-OH of adenosine with a thiol might allow for a covalent bond to form. Since only erbB-1 and erbB-2 have a Cys at this position, the inhibitor should be selective. This model was subsequently tested experimentally by chemical synthesis of 2'-thioadenosine and assayed against the full length erbB-1 receptor and the catalytic domains of erbB-2, insulin receptor, beta-PDGF receptor, and the FGF receptor. Our results show that thioadenosine covalently inactivates erbB-1 with a second-order rate constant of k(max)/K(S) = 2000 +/- 500 M(-1) s(-1). Inactivation is fully reversed by 1 mM dithiothreitol, suggesting that inactivation involves the modification of a cysteine residue at the active site, presumably Cys797. The rate of inactivation saturates with increasing thioadenosine concentrations, suggesting that inactivation occurs through initial formation of a noncovalent complex with K(D) = 1.0 +/- 0.3 microM, followed by the slow formation of a disulfide bond with a rate constant of k(max) = (2.3 +/- 0.2) x 10(-3) s(-1). This approach may have application in the design of selective irreversible inhibitors against other members of the kinase family.

Efficacy of danazol in the control of hormonal migraine.

Of 131 women with hormonally related migraines unresponsive to standard medication, 67 (51.1%) noted profound relief after a 12-month, phased study using danazol for migraine prevention. The first three phases consisted of two-month cycles: dietary control and acetazolamide, the addition of danazol and danazol discontinuation. Eighty-three women (63.36%) reported control of their hormonal migraines while using danazol. In phase IV, 81 women whose headaches were controlled by danazol restarted danazol for an additional six months. Sixty-seven (82.7%) reported continued success with this medication. Danazol proved highly successful in the control of women's cyclic migraine. Its effectiveness remained consistent throughout the treatment course. In the prophylactic treatment of women's hormonal migraine, 400 mg of danazol administered daily for 25 days each month can prove effective when standard medical therapy fails. Furthermore, the response to danazol supported the concept that hormonal migraine should be treated as a distinct clinical entity.

Role of binding energy with coenzyme A in catalysis by 3-oxoacid coenzyme A transferase.

Succinyl-CoA:3-oxoacid coenzyme A transferase (EC 2.8.3.5), which catalyzes the reversible conversion of succinyl-CoA and acetoacetate into acetoacetyl-CoA and succinate through a covalent enzyme thiol ester intermediate, E-CoA, utilizes binding energy from noncovalent interactions with CoA to bring about an increase in kcat/KM of approximately 10(10)-fold. The approximately 40-fold stronger binding of desulfo-CoA (KI = 2.7 +/- 0.7 mM) compared to desulfopantetheine (KI = 110 +/- 15 mM), both of which inhibit competitively with respect to acetoacetyl-CoA, shows that binding to the nucleotide domain of CoA at the active site provides ca. -2.2 kcal/mol of binding energy to stabilize noncovalent complexes with the enzyme. This is much smaller than the ca. -8.9 kcal/mol that the nucleotide domain contributes to the stabilization of the transition state and the ca. -7.2 kcal/mol that it contributes to stabilizing the E-CoA intermediate [Fierke, C. A., & Jencks, W. P. (1986) J. Biol. Chem. 261, 7603-7606]. This shows that most of the approximately 10(6)-fold increase in kcat/KM that is brought about by binding to this domain is in kcat, which is increased by a factor of about 10(5). Binding to the central pantoic acid domain of CoA is stronger in the transition state than in the Michaelis complex by ca. -3.4 kcal/mol; this corresponds to an additional increase in kcat of approximately 350-fold. Covalent enzyme thiol esters analogous to E-CoA but containing the short-chain CoA analogues N-acetylaletheine (NAA) and N-acetylcysteamine (NAC) are more stable than the enzyme thiol ester containing pantetheine (E-Pant) by approximately 3.5 and approximately 4.8 kcal/mol, respectively. Thus, interactions between the pantoic acid domain of CoA and the active site destabilize E-CoA by approximately 4.8 kcal/mol, approximately 1.3 kcal/mol of which arises from interaction with the amide group of the pantoic acid domain and approximately 3.5 kcal/mol of which arises from interaction with other portions of the pantoic acid domain. E-Pant is more reactive toward acetoacetate and succinate by a factor of approximately 10(7) than E-NAA and E-NAC. This shows that the destabilization caused by these interactions in E-CoA is relieved in the transition state, in which binding to the pantoic acid moiety is strongly favorable with delta delta G approximately -5.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous computer-calculated carbon dioxide and oxygen direct Fick and dye dilution measurements of cardiac output in dogs.

Cardiac output was measured by both indocyanine green dye dilution and the direct Fick method using computer-calculated values for oxygen consumption and carbon dioxide excretion in eight mechanically ventilated dogs anesthetized with pentobarbital, with either tubocurarine or succinylcholine intravenous drip for neuromuscular relaxation. Sequential measurements were made during the anesthesia and in response to pharmacologically induced increased cardiac output using doxapram hydrochloride (1.5 mg/kg) given intravenously. The purpose of this project was to investigate the accuracy and reliability of the direct Fick measurements during anesthesia using computer-calculated measurements of pulmonary gas exchange and, since, these measurements have not been reported in detail previously, to establish tentative control values for future projects. The correlation of dye and direct Fick measurements of cardiac output during the first hour after induction of anesthesia was very good (r = 0.85 for all dye-carbon dioxide Fick values; r = 0.83 for all dye-oxygen Fick values.

The structure of human interferon-beta: implications for activity.

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-beta (HuIFN-beta), in particular in relation to its activity. The recently determined crystal structure of HuIFN-beta provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-beta, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-beta.

Dopamine and dobutamine induce hypokalemia in anesthetized dogs.

Epinephrine-induced hypokalemia appears to be mediated by beta 2-agonist activation of Na+/K+ ATPase. To determine whether dopamine and dobutamine induce hypokalemia, eight adult mongrel dogs were anesthetized and studied in random crossover fashion. Potassium [K+] was measured with an ion-selective microelectrode, and central hemodynamics were measured continuously. After stabilization, dopamine and dobutamine were infused at doses of 2, 4, 8, and 20 micrograms/kg/min (15-min increments/dose), and 0.9% NaCl was infused at equivalent volumes, with a 1-h washout between treatments. The mean change in [K+] at each infusion rate was compared between treatments among dogs with an adequate hemodynamic response. Among dopamine responders (n = 5), [K+] decreased from 3.74 +/- 0.42 mEq/L at baseline to 3.63 +/- 0.51 at 2 micrograms/kg/min (p less than 0.02) and was not significantly different at higher doses. Among dobutamine responders (n = 7), [K+] decreased from 3.52 +/- 0.74 at baseline to 3.31 +/- 0.87 at 8 micrograms/kg/min (p less than 0.02) and 3.25 +/- 0.86 at 20 micrograms/kg/min (p less than 0.02), and was not significantly different at lower doses. We conclude that dopamine and dobutamine induce significant hypokalemia, consistent with their adrenergic agonist activity, and this may be related to the known arrhythmogenicity of these agents.

The confirmation of a biochemical marker for women's hormonal migraine: the depo-estradiol challenge test.

PRECIS: Will estrogen withdrawal cause migraines in post-menopausal women? OBJECTIVE: To record the changes in serum estradiol and total estrogen levels after an intramuscular estradiol injection in menopausal subjects and then record any subsequent migraine occurrence. DESIGN: Open selection process, comparative trial. PATIENTS: Twenty-eight postmenopausal women volunteers were given 5 mg depo-estradiol cyprionate as an intramuscular injection. Sixteen (migraine group) had a history of severe, cyclic, menstrually related migraine attacks before becoming menopausal. Twelve (control group) had no history of migraine or headache. All volunteers were on continuous estrogen replacement therapy at the beginning of the study. Progestins were not used in the study. MAIN OUTCOME MEASURES: Serum estradiol and total estrogen levels were measured prior to the depo-estradiol injection and on subsequent days 4, 7, 14, 21, and 28. RESULTS: Total estrogen and estradiol levels varied greatly at every measured interval. Menopausal complaints of vasomotor symptoms were relieved for at least the first 2 weeks of the study. No member of the control group reported a migraine during the month. However, a severe migraine was reported by all 16 women with a history of migraine. The average day of the migraine occurrence was 18.5 +/- 2.8. The serum level of estradiol on the day of the worst migraine was 46.4 +/- 5.6 pg/mL. The significance of these findings was at the 95% confidence level. CONCLUSIONS: This study confirms two factors about menopausal hormonal migraine: (1) it can be precipitated by a drop in serum estrogen levels, and (2) a period of estrogen priming is a necessary prerequisite. This study also identifies that there are two biologically different populations of postmenopausal women: (1) those who developed migraine after a single depo-estradiol injection, and (2) those who did not. By understanding that in addition to the biological predisposition to migraine there exists the biochemical cofactor of falling estrogen levels, we may better understand this phenomenon and develop means to prevent its occurrence.

Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1.

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.

Low affinity binding of an LFA-3/IgG1 fusion protein to CD2+ T cells is independent of cell activation.

Quantitative analysis of binding of the bivalent recombinant soluble fusion protein, LFA-3/IgG1, shows that the fusion protein binds to human CD2+ PBLs primarily through low affinity (KD approximately 140 microM) but also through high avidity (90 nM) interactions. The concentration dependence for LFA-3/IgG1 PBL binding took the form of two overlapping bell-shaped curves separated by a clear and reproducible minimum. This was accounted for in part by minor heterogeneity in the LFA-3/IgG1 preparations, and potentially by the ability of the ligand to bind to both CD2 and Fc receptors (FcR), best evidenced by the distinct binding properties of the fusion protein to NK and T cells. The low affinity LFA-3/ IgG1 binding to T cells is consistent with binding to CD2 only, and is in agreement with the low affinity reported for interactions between soluble forms of LFA-3 and CD2 by surface plasmon resonance technology. Moreover, as the low affinity determinations are similar for CD2 on resting and activated T cells, although the CD2 molecule has been reported to be altered to reveal new epitopes upon T cell activation, the binding data argue against multiple cell activation-dependent affinity states of CD2 for LFA-3 binding. This is distinct from that observed with other adhesion partners, and suggests that the different adhesion pathways utilize distinct mechanisms to mediate cell adhesion.

Noninvasive measurement of pulmonary gas exchange during general anesthesia.

Expired gas flow volume (VE), carbon dioxide excretion (Vco2) and oxygen consumption (Vo2) were measured continuously for 2-minute periods at 15-minute intervals during at least 75 minutes of general anesthesia and surgery in clinical patients. Analog tape-recorded outputs from an infrared CO2 analyzer, from a rapid polarographic O2 analyzer, and from a pneumotachograph were subsequently processed by a general purpose digital computer. Values for VE, VCO2, and VO2 in a group of 50 normal paralyzed endotracheally intubated women with balanced N2O-O2-fentanyl anesthesia for lower abdominal surgery compare favorably with the few published reports of similar measurements. The measured response to anesthesia and surgery in most patients included a progressive increase in O2 uptake and a concurrent but not necessarily simultaneous decrease in CO2 output with a consequent decreased respiratory gas exchange ratio (RE).

Multiple activation states of integrin alpha4beta1 detected through their different affinities for a small molecule ligand.

We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.

Breath-by-breath measurement of oxygen consumption and FIO2-FEO2 with increased oxygen demand.

Continuous on-line breath-by-breath measurement of pulmonary gas exchange was used to monitor the increase in oxygen uptake (VO2) and carbon dioxide excretion (VCO2) induced by the oxidative phosphorylation uncoupling agent 2, 4-dinitrophenol (DNP) in 10 dogs. With incremental doses of DNP totaling 5 mg/kg, the continuously monitored VO2 increased within 2-3 min after the first injection of the drug. VCO2 showed a similar response 4-6 min after the first injection. Temperature increase due to the pharmacological oxidative phosphorylation uncoupling required 20-30 min for a discernible change at this dose. This study also demonstrated a modified and compromised response to the drug in dogs where oxygen delivery was limited by mechanical ventilation.

A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-beta 1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.