Discovery and SAR of pyrrolo[2,1-f][1,2,4]triazin-4-amines as potent and selective PI3Kδ inhibitors.

Aberrant Class I PI3K signaling is a key factor contributing to many immunological disorders and cancers. We have identified 4-amino pyrrolotriazine as a novel chemotype that selectively inhibits PI3Kδ signaling despite not binding to the specificity pocket of PI3Kδ isoform. Structure activity relationship (SAR) led to the identification of compound 30 that demonstrated efficacy in mouse Keyhole Limpet Hemocyanin (KLH) and collagen induced arthritis (CIA) models.

Identification of small molecules that selectively inhibit diacylglycerol lipase-α activity.

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.

Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α.

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.

Cofactor-specific modulation of 11beta-hydroxysteroid dehydrogenase 1 inhibitor potency.

11beta-hydroxysteroid dehydrogenase 1 regulates the tissue availability of cortisol by interconverting cortisone and cortisol. It is capable of functioning as both a reductase and a dehydrogenase depending upon the surrounding milieu. In this work, we have studied the reaction mechanism of a soluble form of human 11beta-hydroxysteroid dehydrogenase 1 and its mode of inhibition by potent and selective inhibitors belonging to three different structural classes. We found that catalysis follows an ordered addition with NADP(H) binding preceding the binding of the steroid. While all three inhibitors tested bound to the steroid binding pocket, they differed in their interactions with the cofactor NADP(H). Compound A, a pyridyl amide bound more efficiently to the NADPH-bound form of 11beta-hydroxysteroid dehydrogenase 1. Compound B, an adamantyl triazole, was unaffected by NADP(H) binding and the sulfonamide, Compound C, showed preferential binding to the NADP+ -bound form of 11beta-hydroxysteroid dehydrogenase 1. These differences were found to augment significant selectivity towards inhibition of the reductase reaction versus the dehydrogenase reaction. This selectivity may translate to differences in the in vivo effects of 11beta-hydroxysteroid dehydrogenase 1 inhibitors.

Feedback activation of factor XI by thrombin does not occur in plasma.

In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombin- or tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity.

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.

Substrate-dependent modulation of the mechanism of factor XIa inhibition.

Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.

CD32-dependent platelet activation by a drug-dependent antibody to glycoprotein IIb/IIIa antagonists.

Thrombocytopenia is observed with a frequency of up to 2% in patients treated with glycoprotein (GP) IIb/IIIa antagonists. We recently provided evidence that thrombocytopenia is caused by antibody binding to drug-induced conformational changes in GP IIb/IIIa. Here, we report that a murine monoclonal antibody binds to GP IIb/IIIa in an antagonist-dependent manner and activates platelets. Platelet stimulation is associated with a disruption of the phospholipid asymmetry, resulting in the assembly of catalytic active intrinsic Xase and prothrombinase complexes. Further mechanistic studies revealed that this response is (I) mediated in cis, (II) not associated with the formation of prothrombotic microparticles, and (III) requires intact platelet signaling and (IV) is blocked by increases in cAMP. The prothrombotic response is not observed using F(ab')2 fragments and is blocked by incubation of platelets with neutralizing antibodies to the platelet FcgammaRIIa receptor (CD 32).Taken together, these observations suggest that GPIIb/IIIa antagonist-dependent antibody binding to the platelet fibrinogen receptor has the propensity to lead to CD32-mediated platelet activation and accelerated platelet clearance, leading to thrombocytopenia.

Prospective testing for drug-dependent antibodies reduces the incidence of thrombocytopenia observed with the small molecule glycoprotein IIb/IIIa antagonist roxifiban: implications for the etiology of thrombocytopenia.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

Regulation of clot retraction by glycoprotein IIb/IIIa antagonists.

Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa induces clot retraction. Significant differences among GP IIb/IIIa antagonists were previously noted to inhibit thromboelastography in whole blood specimens. The relationship between efficacy of these agents and inhibition of clot retraction is unclear. Here, we use a plasma-free clot retraction assay to evaluate potency of GP IIb/IIIa antagonists to inhibit clot retraction and modulate platelet signaling, and to address whether these effects are realized in the clinically relevant dose range. The potencies for inhibition of clot retraction and aggregation are similar for antagonists with high affinity for resting platelets and slow off-rates, whereas lower affinity and fast off-rate antagonists are disproportionately less effective in blocking clot retraction. A positive correlation is observed between inhibition of clot retraction and inhibition of tyrosine dephosphorylation across a number of GP IIb/IIIa antagonist pharmacophores. For lower affinity and fast off-rate antagonists, the concentrations required for inhibition of clot retraction clearly exceed the clinical dose range. Site occupancy studies combined with clot retraction experiments addressed whether high affinity and slow off-rate compounds can alter clot retraction during the dosing interval. Binding studies using [3H] Roxifiban, a high affinity GP IIb/IIIa antagonist, indicate that occupancy of >95% of GP IIb/IIIa sites is required to inhibit clot retraction. This level of occupancy is not routinely achieved in the clinic and is not tolerated, at least for chronic therapy. These results suggest that inhibition of clot retraction is not necessary for efficacy of GP IIb/IIIa antagonists.