Challenges and Hurdles to Business as Usual in Drug Development for Treatment of Rare Diseases.

Only 10-15 first-in-class new medicines are approved each year by the global pharmaceutical industry for all diseases, of which less than a third is for rare (orphan) diseases. The drug discovery processes to identify rare and common diseases are similar, suggesting it will be impossible to discover new drugs for even a small fraction of the rare diseases using the current paradigm. Different approaches are required to address this large unmet medical need.

Phenotypic vs. target-based drug discovery for first-in-class medicines.

Current drug discovery strategies include both molecular and empirical approaches. The molecular approaches are predominantly hypothesis-driven and are referred to as target-based. The empirical approaches are referred to as phenotypic because they rely on phenotypic measures of response. A recent analysis revealed the phenotypic approaches to be the more successful strategy for small-molecule, first-in-class medicines. The rationalization for this success was the unbiased identification of the molecular mechanism of action (MMOA).

Targeting protein ubiquitination for drug discovery. What is in the drug discovery toolbox?

Protein ubiquitination regulates the half-lives of many proteins by targeting them for degradation. Ubiquitination is a specific process associated with several highly regulated biological outcomes including cell cycle progression, differentiation, antigen presentation, retrovirus assembly, apoptosis, signal transduction, transcriptional activation, biological clocks, receptor downregulation and endocytosis. Newly discovered families of ubiquitination and deubiquitination enzymes participate in these processes. These enzymes could provide new families of drug targets and new ways of intervention in many human diseases; however, much work is required to validate this approach. This review will discuss what is in the drug discovery toolbox to assist in the validation of ubiquitination enzymes as therapeutic targets.

Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases.

We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is "homologous to E6AP C-terminus"). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20-100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.

The dynamics of prostaglandin H synthases. Studies with prostaglandin h synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation.

Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidonic acid to prostaglandins. In this report, we describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition. Tyr355 is part of a hydrogen-bonding network located at the entrance to the cyclooxygenase active site. The Y355F mutant exhibited allosteric activation kinetics in the presence of arachidonic acid that was defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1.36 +/- 0.05. Arachidonic acid-induced allosteric activation has not been directly observed with wild type PGHS2. The mutation also decreased the observed time-dependent inhibition by indomethacin, flurbiprofen, RS-57067, and SC-57666. Detailed kinetic analysis showed that the Y355F mutation decreased the transition state energy associated with slow-binding inhibition (EIdouble dagger) relative to the energy associated with catalysis (ESdouble dagger) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067. These observations show Tyr355 to be involved in the molecular mechanism of time-dependent inhibition. We interpret these results to indicate that slow binding inhibitors and the Y355F mutant slow the rate and unmask intrinsic, dynamic events associated with product formation. We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the cyclooxygenase active site. It is these rearrangements that control the rate of substrate binding and ultimately the rate of prostaglandin formation.

Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Gap-filling and end-of-sentence effects in real-time language processing: implications for modeling sentence comprehension in aphasia.

We present an on-line study showing different sources of lexical activation during sentence comprehension, distinguishing in this respect between reflexive syntactic and less temporarily constrained nonsyntactic sources. Specifically, we show that both the syntactic process of gap filling and a nonsyntactic end-of-sentence effect can be measurable in real time and can be temporally separated. The distinction between activation sources provides a new perspective on real-time sentence comprehension in aphasia and accounts for the disparate results reported in the literature.

Differential allosteric regulation of prostaglandin H synthase 1 and 2 by arachidonic acid.

Prostaglandins are synthesized by prostaglandin H synthase (PGHS) 1 and 2. PGHS2 is regulated through inducible expression. We report here the regulation of PGHS1 activity by substrate-dependent cooperative activation. The cooperativity is characterized by a Hill coefficient of 1.29 +/- 0.06, a curved Eadie-Scatchard plot, and activation by low concentrations of competitive inhibitors. The activation also appears to induce a conformational change in the cyclooxygenase site. The cooperativity produces a 2-4-fold greater rate of PGHS2-dependent prostaglandin formation compared with PGHS1-dependent prostaglandin formation at arachidonic acid concentrations below 0.5 microM. A consequence of the PGHS1 cooperativity is that the affinity of many cyclooxygenase inhibitors for PGHS1 decreases in parallel to the activation by arachidonic acid. In contrast, the affinity of these inhibitors for PGHS2 is unaffected by the changes in arachidonic acid concentration. This results in a dramatic difference in PGHS2/PGHS1 selectivity at different arachidonic acid concentrations.

The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen.

We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin. The KI values associated with the formation of the first reversible complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53 +/- 0. 06 and 0.61 +/- 0.08 microM, respectively; the rate constants for the forward isomerization, k2, into the second reversible complex (EI*) were 0.97 +/- 0.99 and 0.11 +/- 0.01 s-1, respectively, and rates of the reverse isomerization from EI*, k-2, were 0.031 +/- 0.004 and 0.0082 +/- 0.0008 s-1, respectively. Indomethacin was estimated to form the EI complex with the same affinity for both PGHS1 and PGHS2, 10.0 +/- 2.8 microM and 11.2 +/- 2.0 microM, respectively, and dissociate from EI* at approximately the same rate 0.0011 +/- 0.0002 s-1 and 0.0031 +/- 0.0003 s-1, respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 +/- 0.08 s-1 as compared with 0.034 +/- 0.004 s-1. These results show that the overall affinity for the inhibition of PGHS1 versus PGHS2 was 30-fold greater for indomethacin (KI* = 0.032 +/- 0.005 and 1.02 +/- 0.08 microM, respectively) and 3-fold greater for flurbiprofen (KI* = 0.017 +/- 0.002 and 0.045 +/- 0.004 microM, respectively). The results also show that for both PGHS1 and PGHS2, flurbiprofen was bound tighter to the initial EI complex than indomethacin; however, the rate of dissociation from EI* was slower for indomethacin than flurbiprofen. The rate of the forward isomerization to EI* is primarily responsible for the selectivity of both NSAIDs for PGHS1. This analysis shows the quantitative importance of the different kinetic parameters upon the overall binding affinity of these NSAIDs and should greatly assist in our understanding of the structural interactions that promote enzyme-inhibitor binding.

Coreference processing and levels of analysis in object-relative constructions; demonstration of antecedent reactivation with the cross-modal priming paradigm.

This paper is concerned with two related issues in sentence processing--one methodological and one theoretical. Methodologically, it provides an unconfounded test of the ability of the cross-modal lexical priming task, when used appropriately, to provide detailed evidence about the time-course of antecedent reactivation during sentence processing. Theoretically, it provides a study of the nature of the representation that is examined when a reference-seeking element is linked to its antecedent during the processing of object-relative clause constructions. In these studies, subjects heard sentences which contained a lexical ambiguity placed in a strong biasing context. In one study this ambiguous word was the "moved" or "fronted" object of the verb in an object-relative construction. A cross-modal lexical priming (CMLP) naming task was used to determine whether one or more of the meanings of the ambiguity are activated at three temporally distinct points during the sentence: (1) immediately after the lexical ambiguity (Study 1); (2) a later point that was 700 milliseconds before the offset of the main verb (Study 2); (3) immediately after this main verb (at the gap in this filler-gap construction) (Study 2). The probes in the CMLP task were controlled for potential confounds. The results demonstrate the following: At Test Point 1, all meanings of the ambiguity were activated; at Test Point 2, neither meaning of the ambiguity was (still) activated; at Test Point 3, only a single (context-relevant) meaning of the ambiguity was reactivated. It is concluded that an underlying (deep; non-surface-level) memorial representation of the sentence is examined during the process of linking an antecedent to a structural position requiring a referent, and that the CMLP task provides an unbiased measure of this reactivation. Further, it is concluded that this effect cannot be accounted for under a "compound cue" (Ratcliff & McKoon, 1994) explanation.

Neurological distribution of processing resources underlying language comprehension.

Using a cross-modal lexical priming technique we provide an on-line examination of the ability of aphasic patients to construct syntactically licensed dependencies in real time. We show a distinct difference between Wernicke's and Broca's aphasic patients with respect to this form of syntactic processing: the Wernicke's patients link the elements of dependency relations in the same manner as do neurologically intact individuals; the Broca's patients show no evidence of such linkage. These findings indicate that the cerebral tissue implicated in Wernicke's aphasia is not crucial for recovering syntactically licensed structural dependencies, while that implicated in Broca's aphasia is. Moreover, additional considerations suggest that the latter region is not the locus of syntactic representations per se, but rather provides the resources that sustain the normal operating characteristics of the lexical processing system-characteristics that are, in turn, necessary for building syntactic representations in real time.

Participant roles and the processing of verbs during sentence comprehension.

This paper explores the nature of thematic information made available when a verb is accessed during sentence comprehension. Following Shapiro, Zurif, and Grimshaw (1987), a cross-modal lexical decision (interference) task was employed to examine whether either the number of argument structures or the number of participant (thematic) roles inherent in a verb cause an increase in processing load upon access of the verb. It was determined that there was no evidence for such an increased processing load covarying with the number of argument structures of the verb, at least for those verb types examined in this study. However, there was an increase in processing load as a direct function of the number of participant roles carried by the verb. It is concluded that the participant roles (thematic roles associated with the central meaning of the verb) are stored with the representation of a verb and are made immediately available upon access of the verb for further processing during comprehension.

Syntactic processing in aphasia.

In this report we comment upon subject selection and methodology, and we describe some recent studies of syntactic processing in aphasia. Our data show that, like neurologically intact subjects, Wernicke's patients reactivate moved constituents (instantiate coreference) at the site of their extraction (even for sentences that they do not understand). Broca's patients, by constrast, are shown not to create such syntactically governed links (even for sentences that they do understand). These data isolate the processing bottleneck in Broca's aphasia and more generally suggest that syntactic comprehension limitations can be traced to changes in cortically localizable resources that sustain lexical processing.

Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase.

Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs.

Azalanstat (RS-21607), a lanosterol 14 alpha-demethylase inhibitor with cholesterol-lowering activity.

Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14 alpha-demethylase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dose-dependent manner (ED50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering (r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50-75 mg/kg) stimulated hepatic microsomal cholesterol 7 alpha-hydroxylase activity by 50-400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis.

The allocation of memory resources during sentence comprehension: evidence from the elderly.

Two experiments were carried out to examine the ability of elderly subjects to establish syntactically governed dependency relations during the course of sentence comprehension. The findings reveal the manner in which memory constraints operate during syntactic processing.

Functional localization in the brain with respect to syntactic processing.

This report describes some recent examinations of the ability of aphasic patients to construct syntactically governed dependency relations in real time. The data show that Wernicke's patients can link the elements of dependency relations in the same way as neurologically intact subjects, even for sentences that they do not understand. Broca's patients, by contrast, are shown to be unable to create such links, even for sentences that they do understand. These data underline the isolability of this stage of syntactic analysis and they suggest that comprehension limitations statable in syntactic terms can be traced to changes in cortically localizable processing resources.