Inhibitors of HIV-1 Nef-Mediated Activation of the Myeloid Src-Family Kinase Hck Block HIV-1 Replication in Macrophages and Disrupt MHC-I Downregulation.

HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730 000 compounds using the Nef·Hck assay identified six unique hit compounds that bound directly to recombinant Nef by surface plasmon resonance (SPR) in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 µM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the µ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.

Identification of DOT1L inhibitors by structure-based virtual screening adapted from a nucleoside-focused library.

Disruptor of Telomeric Silencing 1-Like (DOT1L), the sole histone H3 lysine 79 (H3K79) methyltransferase, is required for leukemogenic transformation in a subset of leukemias bearing chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene, as well as other cancers. Thus, DOT1L is an attractive therapeutic target and discovery of small molecule inhibitors remain of high interest. Herein, we are presenting screening results for a unique focused library of 1200 nucleoside analogs originally produced under the aegis of the NIH Pilot Scale Library Program. The complete nucleoside set was screened virtually against DOT1L, resulting in 210 putative hits. In vitro screening of the virtual hits resulted in validation of 11 compounds as DOT1L inhibitors clustered into two distinct chemical classes, adenosine-based inhibitors and a new chemotype that lacks adenosine. Based on the developed DOT1L ligand binding model, a structure-based design strategy was applied and a second-generation of non-nucleoside DOT1L inhibitors was developed. Newly synthesized compound 25 was the most potent DOT1L inhibitor in the new series with an IC50 of 1.0 µM, showing 40-fold improvement in comparison with hit 9 and exhibiting reasonable on target effects in a DOT1L dependent murine cell line. These compounds represent novel chemical probes with a unique non-nucleoside scaffold that bind and compete with the SAM binding site of DOT1L, thus providing foundation for further medicinal chemistry efforts to develop more potent compounds.

A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.

The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition.

The G2019S mutation in LRRK2 is one of the most common known genetic causes of neurodegeneration and Parkinson disease (PD). LRRK2 mutations are thought to enhance LRRK2 kinase activity. Efficacious small molecule LRRK2 kinase inhibitors with favorable drug properties have recently been developed for pre-clinical studies in rodent models, and inhibitors have advanced to safety trials in humans. Rats that express human G2019S-LRRK2 protein and G2019S-LRRK2 knock-in mice provide newly characterized models to better understand the ostensible target for inhibitors. Herein, we explore the relationships between LRRK2 kinase inhibition in the brain and the periphery to establish the link between LRRK2 kinase activity and protein stability, induction of lysosomal defects in kidney and lung, and how G2019S-LRRK2 expression impacts these phenotypes. Using a novel ultra-sensitive scalable assay based on protein capillary electrophoresis with LRRK2 kinase inhibitors included in-diet, G2019S-LRRK2 protein was resilient to inhibition compared to wild-type (WT)-LRRK2 protein, particularly in the brain. Whereas WT-LRRK2 kinase activity could be completed blocked without lowering LRRK2 protein levels, higher inhibitor concentrations were necessary to fully reduce G2019S-LRRK2 activity. G2019S-LRRK2 expression afforded robust protection from inhibitor-induced kidney lysosomal defects, suggesting a gain-of-function for the mutation in this phenotype. In rodents treated with inhibitors, parallel measurements of phospho-Rab10 revealed a poor correlation to phospho-LRRK2, likely due to cells that express Rab10 but poorly express LRRK2 in heterogenous tissues and cell isolates. In summary, our results highlight several challenges associated with the inhibition of the G2019S-LRRK2 kinase that might be considered in initial clinical efforts.

Identification of quinazoline compounds as novel potent inhibitors of Wnt/ß-catenin signaling in colorectal cancer cells.

The Wnt/ß-catenin signaling pathway is critical for the initiation and progression of most colon cancers, and has emerged as one of the most promising targets for colorectal cancer chemoprevention and treatment. In this study, we have discovered a structurally related series of quinazolines as potent inhibitors of Wnt/ß-catenin signaling in colorectal cancer cells harboring mutations in CTNNB1 or APC. We showed that the quinazoline leads suppressed Wnt/ß-catenin signaling without altering the level of ß-catenin protein in colorectal cancer cells, suggesting that they act on the downstream elements of the pathway. Moreover, the quinazoline leads displayed potent anticancer activities with IC50 values between 4.9 and 17.4 µM in colorectal cancer cells. Importantly, we also found that a structurally related quinazoline lacking inhibitory effect on Wnt/ß-catenin signaling was unable to suppress colorectal cancer cell proliferation. Together, these results suggest that the quinazoline lead compounds identified in this study have therapeutic potential for the prevention and treatment of colorectal cancer.

Synthesis and general biological activity of a small adenosine-5'-(carboxamide and sulfanilamide) library.

A small library of fifty-five adenosine peptide analogs was synthesized, under the Pilot Scale Library (PSL) Program of the NIH Roadmap initiative, from 2',3'-O-isopropylideneadenosine-5'-carboxylic acid 2. The coupling of amine or sulfanilamide reactants to the free 5'-carboxylic acid moiety of 2, in automated solution-phase fashion, led after acid-mediated hydrolysis to target compounds 3-57 in good yields and high purity. No marked anticancer or antimalarial activity was noted on preliminary cellular testing. Initial screening through the MLPCN program, however, indicates that these analogs may show diverse and interesting biological activities.

Unique functional and structural properties of the LRRK2 protein ATP-binding pocket.

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.

Parallel solution-phase synthesis and general biological activity of a uridine antibiotic analog library.

A small library of ninety four uridine antibiotic analogs was synthesized, under the Pilot Scale Library (PSL) Program of the NIH Roadmap initiative, from amine 2 and carboxylic acids 33 and 77 in solution-phase fashion. Diverse aldehyde, sulfonyl chloride, and carboxylic acid reactant sets were condensed to 2, leading after acid-mediated hydrolysis, to the targeted compounds 3-32 in good yields and high purity. Similarly, treatment of 33 with diverse amines and sulfonamides gave 34-75. The coupling of the amino terminus of d-phenylalanine methyl ester to the free 5'-carboxylic acid moiety of 33 followed by sodium hydroxide treatment led to carboxylic acid analog 77. Hydrolysis of this material gave analog 78. The intermediate 77 served as the precursor for the preparation of novel dipeptidyl uridine analogs 79-99 through peptide coupling reactions to diverse amine reactants. None of the described compounds show significant anticancer or antimalarial acivity. A number of samples exhibited a variety of promising inhibitory, agonist, antagonist, or activator properties with enzymes and receptors in primary screens supplied and reported through the NIH MLPCN program.

Synthesis of novel peptidyl adenosine antibiotic analogs.

A small library of peptidyl adenosine antibiotic analogs was synthesized, under the Pilot Scale Library Program of the NIH Roadmap initiative, from 2',3'-O-isoproylideneadenosine-5'-carboxylic acid 2 in excellent yield. The coupling of the amino terminus of L-2-aminophenylbutyric methyl ester to a free 5'-carboxylic acid moiety of 2 followed by sodium hydroxide treatment led to carboxylic acid analog 4. Hydrolysis of this latter gave unprotected nucleoside analog 5. Intermediate 4 served as the precursor for the preparation of novel peptidyl adenosine analogs 6-18 in good yields and high purity through peptide coupling reactions to diverse amine derivatives. No marked anticancer and antimalaria activity was noted on preliminary cellular testing; however these analogs should be useful candidates for other types of biological activity.

Parallel solution-phase synthesis of an adenosine antibiotic analog library.

A library of eighty one adenosine antibiotic analogs was prepared under the Pilot Scale Library Program of the NIH Roadmap initiative from 5'-amino-5'-deoxy-2',3'-O-isopropylidene-adenosine 3. Diverse aldehyde, sulfonyl chloride and carboxylic acid reactant sets were condensed to 3, in solution-phase fashion, leading after acid-mediated hydrolysis to the targeted compounds in good yields and high purity. No marked antituberculosis or anticancer activity was noted on preliminary cellular testing, but these nucleoside analogs should be useful candidates for other types of biological activity.

Structure-activity relationship for adenosine kinase from Mycobacterium tuberculosis II. Modifications to the ribofuranosyl moiety.

Adenosine kinase (Ado kinase) from Mycobacterium tuberculosis is structurally and biochemically unique from other known Ado kinases. This purine salvage enzyme catalyzes the first step in the conversion of the adenosine analog, 2-methyl-Ado (methyl-Ado), into a metabolite with antitubercular activity. Methyl-Ado has provided proof of concept that the purine salvage pathway from M. tuberculosis may be utilized for the development of antitubercular compounds with novel mechanisms of action. In order to utilize this enzyme, it is necessary to understand the topography of the active site to rationally design compounds that are more potent and selective substrates for Ado kinase. A previous structure-activity relationship identified modifications to the base moiety of adenosine (Ado) that result in substrate and inhibitor activity. In an extension of that work, 62 Ado analogs with modifications to the ribofuranosyl moiety, modifications to the base and ribofuranosyl moiety, or modifications to the glycosidic bond position have been analyzed as substrates and inhibitors of M. tuberculosis Ado kinase. A subset of these compounds was further analyzed in human Ado kinase for the sake of comparison. Although no modifications to the ribose moiety resulted in compounds as active as Ado, the best substrates identified were carbocyclic-Ado, 8-aza-carbocyclic-Ado, and 9-[alpha-l-lyxofuranosyl]-adenine with 38%, 4.3%, and 3.8% of the activity of Ado, respectively. The most potent inhibitor identified, 5'-amino-5'-deoxy-Ado, had a K(i)=0.8muM and a competitive mode of inhibition. MIC studies demonstrated that poor substrates could still have potent antitubercular activity.

Synthesis and biological activity of some 4-substituted 1-[1-(2,3-dihydroxy-1-propoxy)methyl-1,2,3-triazol-(4 & 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines.

Cycloaddition of 7a, b with 6 gave, after separation and deprotection, two regioisomers 10a, b and 11a, b. The deprotected acyclic nucleoside 10a used as the precursor for the preparation of 4-amino (12), 4-methylamino (13), 4-benzylamino (14), 4-methoxy (15) and 4-hydroxy (16) analogues. All acyclic nucleosides were evaluated for their inhibitory effects against HIV-1(IIIB), HIV-2(ROD) in MT-4 cells, for their anti-tumor activity and for their inhibitory effects against Mycobacterium tuberculosis. No marked activity was found.