PURPOSE:In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES:ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS:Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS:[18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.

We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Ki = 22.4 μM), exhibited good binding efficiencies (LBE = 0.49, LLE = 4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.

Alzheimer's disease (AD) patients suffer from an inexorable loss of cognitive function due in part to the degeneration of the basal forebrain cholinergic projection neurons. Restoring signalling through the post-synaptic muscarinic acetylcholine receptors that mediate cholinergic signalling is thus a promising therapeutic strategy for the symptomatic management of AD. Moreover muscarinic receptor agonists reduce Aβ42 in animal models, suggesting that this class of compounds could address the underlying disease pathology. Unfortunately, multiple non-selective muscarinic receptor agonists have been tested in AD patients and were not tolerated due to unwanted peripheral cholinergic stimulation. Improved selectivity for M1, the most prominently expressed post-synaptic muscarinic receptor in the cortex and hippocampus, could address these tolerability issues, however the high conservation of the acetylcholine binding site between receptor subtypes has precluded the discovery of selective agonists. To achieve selectivity for M1 by discovering and exploiting an allosteric ligand binding site. We screened a chemical library for positive allosteric modulators of the M1 receptor and assessed chemical leads for selectivity in vitro and efficacy in rodent AD models in vivo. We identified Benzyl Quinolone Carboxylic Acid (BQCA), a small molecule potentiator selective for M1. In CHO cells expressing recombinant human receptor, BQCA sensitizes M1 to acetylcholine 83.9-fold (inflection point = 449 nM), while having no potentiation, agonist, or antagonist effect on M2, M3, or M4 receptors up to 100 μM. In the mouse contextual fear conditioning model of episodic-like memory, BQCA fully reversed the cognitive impairment caused by the non-selective muscarinic antagonist scopolamine, demonstrating a critical role for M1 in this type of memory. Importantly BQCA does not show signs of unwanted peripheral cholinergic stimulation at doses that produce central physiological responses. In vivo BQCA and other muscarinic agonists can specifically reduce cortical Aβ42 in some experiments. However, this effect is likely complex and indirect, since it does not occur in cultured neurons and is not consistently observed across species and disease models. BQCA is a highly selective pharmacological reagent for understanding the normal physiology of the M1 receptor and its potential as a therapeutic target for AD.

Neuronal Leucine Rich Repeat (LRR) containing proteins have an emerging role in regulating neurite outgrowth and synapse formation. Many of these neuronally expressed LRR proteins are also expressed in regions of the adult brain which undergo neurogenesis and are involved in cognition and memory. Interestingly, the LRR protein, LRRTM3, alters the levels of Abeta peptide secreted from neuronal cells by regulating the amount of beta-site cleaved APP substrate generated inside the cell. Thus, the study of LRRTM3 at a cellular level may help understand biochemical pathways regulated by LRR proteins underlying neurodegenerative processes. We have examined the expression and impact on APP metabolism of 2 identified isoforms of LRRTM3. In rhesus monkey brain the “long” isoform of LRRTM3 is expressed in cortical regions as well as in Layer 3 and 5 neurons of the entorhinal cortex whereas the “short” isoform is restricted to the dentate gyrus (a region of much neurogenesis) in the hippocampus. In a temporal study spanning 3 month to 18 month old tg2576 and wild-type mice, long isoform specific in situ hybridization identified cortical regions strongly expressed in the adult brain throughout the timecourse. In complementary studies using SH-SY5Y cells, isoform selective siRNAs of the long but not short isoform significantly reduce Abeta40 and Abeta42 secretion. In addition, the long isoform which expresses a highly conserved yet unique 68 additional amino acid C-terminal tail is expressed on the cell surface of SH-SY5Y and COS cells. This combination of cellular, imaging and biochemical studies as well as future in vivo studies will help elucidate the role of LRRTM3 in the diseased brain.

A key goal within the Alzheimer's disease (AD) research community is the early clinical detection and diagnosis of AD in patients who are at risk, or present with symptoms, of the disease. In addition to the standard clinical criteria, much effort in the field is aimed at identifying biochemical and imaging biomarkers. In a collaborative effort to discover early biomarkers of AD between OPTIMA and Merck Research Laboratories, we have expanded a panel of six previously validated and commercially available CSF biochemical assays for APP catabolites (Aβ40, Aβ42, sAPPα, sAPPβ) and tau (tTau, pTau-181). Three new assays were developed, optimized and analytically validated for (1) CSF BACE activity, (2) CSF oligomeric Aβ, and (3) CSF Homocysteine (HCY) levels. These biochemical markers were measured in the CSF of pathologically confirmed AD (N=27–30) and clinically evaluated control cases (N=29). Uni variate results exhibited an age-dependent increase in CSF BACE activity (∼1.0 pM/yr, p<0.05). In AD subjects, a modest but significant decline in age-adjusted CSF BACE activity was observed compared to controls (50% reduction, p=0.02). Regarding oligomeric Aβ, in a commercially purchased sample set of human CSF, we found AD patients have 82 % higher oligomeric Aβ levels than their age-matched non-demented controls (p<0.01). When oligomeric Aβ was assessed in the post-mortem confirmed OPTIMA samples, there did not appear to be a significant difference between AD and CTL. One can't be sure if there is an assay sensitivity issue or the demographics of the sample sets were different. Finally regarding CSF Homocysteine, we observed meaningful age dependence in CSF Homocysteine levels (slope ∼ 0.016 per year, p<0.0003). Differences between AD and CTL after age-adjustment are significant in females (11.2% greater in AD v CTL females, p = 0.049). Taken together, these findings obtained from a post-mortem confirmed case analysis, enhance the putative value and use of specific CSF markers in support of the detection and diagnosis of Alzheimer's disease.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2'-C-methyl-adenosine and 2'-O-methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2'-C-methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2'-O-methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2'-O-methyl-cytidine nucleoside to the active 5'-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

Low-density polyethylene film was coated with a solution containing a high-molecular-weight or low-molecular-weight methylcellulose and hydroxypropyl methylcellulose. Films contained 10000, 7500, 5000, 2500, or 0 IU/cm(2) nisin. Film samples were placed into peptone water, and 10-muL samples were removed and placed onto spiral plated lawns of Listeria monocytogenes. Zones of inhibition were measured using a caliper. Films containing 5000, 7500, and 10000 IU/cm(2) nisin inhibited L. monocytogenes after 30 min; films with 7500 and 10000 IU/cm(2) nisin inhibited L. monocytogenes after 60 min and 8 h, respectively. No zones of inhibition were observed after 24 h and 4 d for all films. After 8 d, zones of inhibition were observed for films with a levels of nisin except 2500 IU/cm(2). Films with 0 and 2500 IU/cm(2) nisin did not produce zones of inhibition throughout the study. Molecular weight of the cellulose-based carrier had no effect on inhibition of L. monocytogenes. A standard curve of inhibition was developed using solutions of 10000, 7500, 5000, 2500, or 0 IU/cm(2) nisin applied directly to lawns of L. monocytogenes. The amount of inhibition using a direct application of a solution was 25% to 50% more effective for inhibition of L. monocytogenes compared with the coated film samples. Overall, the coated film samples were effective for inhibition of L. monocytogenes, particularly when 7500 and 10000 IU/cm(2) nisin were used, but the release of nisin was not controlled and did not provide consistent inhibition throughout the 8-d study.

Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.