Purpose Tau deposition is a key pathological feature of Alzheimer’s disease (AD) and other neurodegenerative disorders. The spreading of tau neurofibrillary tangles across defined brain regions corresponds to the observed level of cognitive decline in AD. Positron-emission tomography (PET) has proved to be an important tool for the detection of amyloid-beta (Aβ) aggregates in the brain, and is currently being explored for detection of pathological misfolded tau in AD and other non-AD tauopathies. Several PET tracers targeting tau deposits have been discovered and tested in humans. Limitations have been reported, especially regarding their selectivity. Methods In our screening campaign we identified pyrrolo[2,3- b :4,5- c ’]dipyridine core structures with high affinity for aggregated tau. Further characterization showed that compounds containing this moiety had significantly reduced monoamine oxidase A (MAO-A) binding compared to pyrido[4,3- b ]indole derivatives such as AV-1451. Results Here we present preclinical data of all ten fluoropyridine regioisomers attached to the pyrrolo[2,3- b :4,5- c ’]dipyridine scaffold, revealing compounds 4 and 7 with superior properties. The lead candidate [ 18 F]PI-2620 (compound 7 ) displayed high affinity for tau deposits in AD brain homogenate competition assays. Specific binding to pathological misfolded tau was further demonstrated by autoradiography on AD brain sections (Braak I-VI), Pick’s disease and progressive supranuclear palsy (PSP) pathology, whereas no specific tracer binding was detected on brain slices from non-demented donors. In addition to its high affinity binding to tau aggregates, the compound showed excellent selectivity with no off-target binding to Aβ or MAO-A/B. Good brain uptake and fast washout were observed in healthy mice and non-human primates. Conclusions Therefore, [ 18 F]PI-2620 was selected for clinical validation. Electronic supplementary material The online version of this article (10.1007/s00259-019-04397-2) contains supplementary material, which is available to authorized users.

18 F-PI-2620 is a PET tracer with high binding affinity for aggregated tau, a key pathologic feature of Alzheimer disease (AD) and other neurodegenerative disorders. Preclinically, 18 F-PI-2620 binds to both 3-repeat and 4-repeat tau isoforms. The purpose of this firstin-humans study was to evaluate the ability of 18 F-PI-2620 to detect tau pathology in AD patients using PET imaging, as well as to assess the safety and tolerability of this new tau PET tracer. Methods: Participants with a clinical diagnosis of probable AD and healthy controls (HCs) underwent dynamic 18 F-PI-2620 PET imaging for 180 min. 18 F-PI-2620 binding was assessed visually and quantitatively using distribution volume ratios (DVR) estimated from noninvasive tracer kinetics and SUV ratio (SUVR) measured at different time points after injection, with the cerebellar cortex as the reference region. Time-activity curves and SUVR were assessed in AD and HC subjects, as well as DVR and SUVR correlations and effect size (Cohen's d) over time. Results: 18 F-PI-2620 showed peak brain uptake around 5 min after injection and fast washout from nontarget regions. In AD subjects, focal asymmetric uptake was evident in temporal and parietal lobes, precuneus, and posterior cingulate cortex. DVR and SUVR in these regions were significantly higher in AD subjects than in HCs. Very low background signal was observed in HCs. 18 F-PI-2620 administration was safe and well tolerated. SUVR time-activity curves in most regions and subjects achieved a secular equilibrium after 40 min after injection. A strong correlation (R 2. 0.93) was found between noninvasive DVR and SUVR for all imaging windows starting at more than 30 min after injection. Similar effect sizes between AD and HC groups were obtained across the different imaging windows. 18 F-PI-2620 uptake in neocortical regions significantly correlated with the degree of cognitive impairment. Conclusion: Initial clinical data obtained in AD and HC subjects demonstrated a high image quality and excellent signal-to-noise ratio of 18 F-PI-2620 PET for imaging tau deposition in AD subjects. Noninvasive quantification using DVR and SUVR for 30-min imaging windows between 30 and 90 min after injection-for example, 45-75 min-provides robust and significant discrimination between AD and HC subjects. 18 F-PI-2620 uptake in expected regions correlates strongly with neurocognitive performance.

Objective. Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity.Methods. Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors.Results. A number of non-hydroxamic acidcontaining compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompeti-

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A⅐G-BaP DE-S and -R mismatches over C⅐G-BaP DE-S and -R correct matches by factors of ϳ350-and 130-fold, respectively, even favoring G⅐G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10 ؊2 to 10 ؊4 depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOSinduced ⌬dinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.Recent studies suggest that in addition to DNA polymerases (pol) 1 I-III, which are prototypes of the A-, B-, and C-polymerase families, respectively (1, 2), Escherichia coli possesses two members of the recently described Y-family of DNA polymerases (3). This new polymerase family is typified by the UmuC, DinB, Rev1, and Rad30 proteins, which represent distinct phylogenetic branches of the Y-family tree (4, 5). The Y-family polymerases lack intrinsic exonuclease activity and are distributive in the absence of stimulatory accessory factors (6). Members of this new polymerase family are best characterized by their lesion-bypassing properties; but it is clear that when they replicate undamaged DNA, they do so with fidelities much lower than those of the A-, B-, and C-family polymerases (6).Both E. coli Y-family pol IV (DinB) and pol V (UmuDЈ 2 C) are expressed at elevated levels as part of the cell's global SOS response to DNA damage (7). It is clear from years of genetic characterization of umuD and umuC mutants that the principal role of pol V is to bypass template bases that effectively block normal pol III-catalyzed DNA replication because deletion of the umu operon or missense mutations in either umuD or umuC effectively render E. coli cells non-mutable, despite being exposed to a variety of mutagens (8 -10). Translesion DNA synthesis catalyzed by pol V results in mutations targeted directly opposite DNA damage sites. However, in addition to its primary role in replicating across from damaged bases, pol V also causes untargeted base substitution mutations in the absence of DNA damage (11).pol IV is responsible for generating frameshift mutations on undamaged DNA, causing adaptive mutations in non-divi...

A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a shortlived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The crosslinking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N 2 amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes. HIV-11 reverse transcriptase (RT) is a complex molecular machine that uses several kinetically distinct steps to incorporate a nucleotide into a growing DNA strand. It is a heterodimer composed of a larger 560-residue subunit (p66) and a smaller subunit (p51) that contains the N-terminal 440 residues of p66. Both subunits contain subdomains that were named fingers, palm, thumb, and connection, because of the similarity of p66 to a right hand. The DNA polymerase active site is located in the p66 palm subdomain and the DNA binding cleft is formed primarily by the p66 fingers, palm, and thumb subdomains. The mechanism of polymerization by RT is similar to other polymerases and involves: 1) binding of the DNA substrate to the apo-enzyme; 2) binding of dNTP and divalent metal ions (required for catalysis) to the enzyme⅐DNA complex, followed by rate-limiting conformational changes; 3) formation of a phosphodiester bond between the 3Ј-OH primer terminus and the ␣-phosphate of dNTP, followed by release of the pyrophosphate product; 4) translocation of the elongated DNA primer (for processive synthesis) from the dNTP-binding site (N site) to the priming site (P site) or release of the nucleic acid (distributive synthesis) (Fig. 1).Extensive biochemical and crystallographic studies have enhanced our understanding of the details of the mechanism of DNA polymerization. However, the translocation step rem...

Neuraminidase, a key enzyme responsible for influenza virus propagation, has been used as a template for selective synthesis of small subsets of its own inhibitors from theoretically highly diverse dynamic combinatorial libraries. We show that the library building blocks, aldehydes and amines, form significant amounts of the library components resulting from their coupling by reductive amination only in the presence of the enzyme. The target amplifies the best hits at least 120-fold. The dynamic libraries synthesized and screened in such an in vitro virtual mode form the components that possess high inhibitory activity, as confirmed by enzyme assays with independently synthesized individual compounds.

New and potent inhibitors of neuraminidase, a key enzyme in the influenza virus activity, have been discovered in dynamic combinatorial libraries based on ketones and amines as building blocks. Selective synthesis of a number of inhibitors among multiple theoretically possible combinations of building blocks is driven by the presence of the target enzyme.

Human DNA polymerase was used to copy four stereoisomeric deoxyguanosine (dG) adducts derived from benzo[a]pyrene 7,8-diol 9,10-epoxide (diastereomer with the 7-hydroxyl group and epoxide oxygen trans (BaP DE-2)). The adducts, formed by either cis or trans epoxide ring opening of each enantiomer of BaP DE-2 by N 2 of dG, were placed at the fourth nucleotide from the 5-end in two 16-mer sequence contexts, 5ϳCG*Aϳ and 5ϳGG*T. pol was remarkably error prone at all four diol epoxide adducts, preferring to misincorporate G and A at frequencies 3-to more than 50-fold greater than the frequencies for T or the correct C, although the highest rates were 60-fold below the rate of incorporation of C opposite a non-adducted G. Anti to syn rotation of the adducted base, consistent with previous NMR data for a BaP DE-2 dG adduct placed just beyond a primer terminus, provides a rationale for preferring purine misincorporation. Extension of purine misincorporations occurred preferentially, but extension beyond the adduct site was weak with V max /K m values generally 10-fold less than for misincorporation. Mostly A was incorporated opposite (؉)-BaP DE-2 dG adducts, which correlates with published observations that G 3 T is the most common type of mutation that (؉)-BaP DE-2 induces in mammalian cells.Somatic mutation of proto-oncogenes and tumor suppressor genes is a key component in the initiation of cancer (1). Any DNA damage that escapes repair could lead to mutations. However, replicative DNA polymerases are blocked in a potentially lethal manner when they encounter bulky adducts (2). Thus, they are unable to convert adducts to mutations. Recently, a growing number of DNA polymerases capable of conducting translesion DNA synthesis have been discovered (3-11); these likely hold the key to mutagenesis and the initiation of cancer induced by bulky adducts. One of these lesion-bypassing DNA polymerases, human DNA polymerase eta (pol) 1 (12, 13) is a member of the UmuC/DinB/Rev1/Rad30 superfamily (now called the Y family (14)) of DNA polymerases. In humans it is a product of the XPV skin cancer susceptibility gene; inactivation of pol results in a variant form of xeroderma pigmentosum (12,15). pol incorporates mostly correctly at cis-syn T-T cyclobutane dimers (16,17), N-(deoxyguanosin-8-yl)-acetylaminofluorene (18), and cis-Pt G-G adducts (18,19); small amounts of incorrect nucleotides are also incorporated, mostly at the level seen with undamaged DNA. With (6-4) T-T photoproducts, however, a single G is preferentially incorporated (20). Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens (21). Bay-region diol epoxides, formed on an angular benzo-ring, have been shown to be the ultimate carcinogenic metabolites of the PAHs (21-25). Four optically active bay-region diol epoxide isomers (enantiomers of a pair of diastereomers) are formed metabolically from a given hydrocarbon. In the DE-1 ("syn") diastereomer, the epoxide oxygen and benzylic 7-hydroxyl groups are cis, whereas in the DE-2 ("anti") diast...