Optimization of a screening lead for factor VIIa/TF.
The structure-based design and progression of a screening lead to a 3nM factor VIIa/TF inhibitor with improved selectivity versus related enzymes is described.
Amidines↗
Biomedical subjects
K Elrod
Publications and source records attributed to K Elrod.
Development of potent and selective factor Xa inhibitors.
The development of potent and selective small molecule inhibitors of factor Xa is described.
Amidines↗
Neutral inhibitors of the serine protease factor Xa.
A neutral inhibitor of the serine protease factor Xa was identified via a high-throughput screen of a commercial library. The initial lead 1 demonstrated reversible and competitive inhibition kinetics for factor Xa and possessed a high degree of selectivity versus other related serine proteases. Initial modeling efforts and the generation of a series of analogues of 1 are described.
Aniline Compounds↗
A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site.
We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.
Amino Acid Motifs↗
Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets.
BACKGROUND: Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts. RESULTS: Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle. CONCLUSIONS: Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.
Animals↗
UCB 29120, a novel, potential psychotropic agent, alters norepinephrine and dopamine content of rat brain.
UCB 29120 belongs to a novel family of compounds possessing interesting behavioral and physiological properties. Behavioral studies in the rat have revealed the ability of the compound to inhibit scopolamine-induced amnesia while physiological studies demonstrated a significant drug-induced hypothermic response and increase corticosterone plasma levels following acute administration of the compound. In the present study we examined the time-course effects of acute administration of UCB 29120 on levels of catecholamines (norepinephrine, NE; dopamine, DA), indoleamines (serotonin, 5-HT) and metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; 5-hydroxyindoleacetic acid, 5-HIAA) in the rat hypothalamus. Hippocampal, septal and striatal tissue content of the same were also examined at the longest time point employed. In the hypothalamus, UCB 29120 induced significant decreases in NE content 30 min following administration which persisted for at least an additional 30 min, while significant increases in DA and/or DOPAC (and the DOPAC/DA ratio) were measured as early as 5 min following administration and persisted through at least a total of 120 min. Similar, significant changes in dopaminergic parameters were also evident in the other three brain regions at 120 min post-administration. No significant alterations in hypothalamic 5-HT or 5-HIAA were measured at any time point. Acute administration of UCB 29120 may selectively influence catecholaminergic neurotransmitter systems in rat brain.
3,4-Dihydroxyphenylacetic Acid↗
Correlation of the amnestic effects of nicotinic antagonists with inhibition of regional brain acetylcholine synthesis in rats.
Acetylcholine (ACh) is considered to play a primary role in normal mnemonic functioning. Although most research has centered on the central muscarinic cholinergic system, of recent interest in normal and pathologic cognitive processing is the role of the nicotinic cholinergic system. The purpose of this research was to further characterize the role of the nicotinic system in learning and memory processing in a classical passive avoidance task by rats. The centrally acting nicotinic antagonist mecamylamine produced a dose-dependent impairment of performance in the passive avoidance task, however, the peripherally acting nicotinic antagonist hexamethonium was ineffective in this test. To elucidate the potential neurochemical mechanisms underlying mecamylamine's amnestic effect, we examined the ability of the drug to alter specific dynamic components of rat brain cholinergic systems in five brain regions. Mecamylamine produced a dose-dependent inhibition of the synthesis of [3H]ACh from pulse-injected [3H]choline in all regions examined, but was without effect on endogenous, steady-state levels of ACh. As with the behavioral study, administration of hexamethonium was without effect on [3H]ACh synthesis. Calculation of the turnover rates of ACh allowed a correlation between the inhibition of the behavioral task and the inhibition of central cholinergic function. The high degree of correlation obtained (as well as earlier studies in nonhuman primates) underlined the possibility that presynaptically located nicotinic receptors on brain cholinergic neurons are tonically active and mediate a positive feedback mechanism for controlling cholinergic neuronal activity. These receptors may be the neurochemical substrate which underlie the behavioral changes after administration of nicotinic agonists and antagonists.
Acetylcholine↗
Nicotine enhances delayed matching-to-sample performance by primates.
The non-human primate provides an excellent model for studies of learning and memory, and one particular test, the delayed matching-to-sample task, is performed in a similar manner by both humans and non-human primates. Five young adult macaques were employed in this study, displaying variable capacities for retention in the task. Baseline performance was very consistent and three levels of performance difficulties (95-100%, 80-85% and 65-75% correct choices) were employed by including several delay intervals (0-60 sec) in each session. A reproducible enhancement in performance by nicotine in macaques performing a delayed matching-to-sample task was demonstrated. Nicotine enhanced performance with an average increase of 10% at the longest retention delay interval. This beneficial effect of nicotine was abolished in animals pretreated with a low dose (0.5 mg/kg) of mecamylamine to block central nicotinic receptors. Selective blockade of peripheral nicotinic receptors with hexamethonium was without effect on the nicotine response. A high dose (2 mg/kg) of mecamylamine itself induced a marked inhibition of performance, while an equivalent dose of hexamethonium was without effect. These experiments point to the possibility that central nicotinic receptors may be exploited pharmacologically to enhance memory performance. In this respect it is interesting that nicotine was most effective at enhancing performance when recall was more difficult, that is, on the longer retention interval delays. This could signify that nicotine might be particularly effective in the most impaired individuals. Lastly, it is encouraging that the mecamylamine induced decrease in cognitive performance might provide a new model of memory impairment from which to study the pathogenesis and develop new pharmacological strategies for the dementias.
Animals↗
An evaluation of the mechanism of scopolamine-induced impairment in two passive avoidance protocols.
The effects of several doses of the centrally-acting muscarinic antagonist, scopolamine, on subsequent learning and memory were examined using two versions of a standard light versus dark passive avoidance paradigm. The first protocol was employed primarily to examine the effects of scopolamine on the acquisition component of learning and memory as subject performance was measured during five successive (repeated) training trials. The second protocol employed a one-trial twenty-four hour retention task in which subjects were given one training trial followed by one testing trial twenty-four hours later. This latter test encompasses acquisition, retention, and recall components of learning and memory. Dose response studies indicated an effective dose range of 0.4-1.2 mg/kg with 0.8 mg/kg producing maximal performance decrement. Differential scopolamine treatment demonstrated that the drug's primary effect was on the acquisition component only under the present experimental protocols. Furthermore, scopolamine was not found to produce state-dependent learning. Animals administered scopolamine before training and testing failed to perform better than animals receiving pre-training administration only.
Animals↗
Inactivation of kallikrein and kininases and stabilization of whole rat brain kinin levels following focused microwave irradiation.
Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radioimmunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen.
Animals↗