High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.

The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

Programs to facilitate tuberculosis drug discovery: the tuberculosis antimicrobial acquisition and coordinating facility.

There is a real need to discover new drugs that are active on drug-resistant tuberculosis (TB), and for drugs that will shorten the time of therapy. Large pharmaceutical companies have traditionally led the quest for discovering and developing new antiinfective agents but this is not the case when it comes to diseases like tuberculosis that primarily occur in resource restricted countries. Throughout the world many research groups are actively engaged in the scientific discovery of new TB drugs. Unfortunately, most research laboratories do not have the necessary safety facilities or resources for all facets of TB drug discovery. The Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) was established in order to make comprehensive testing services available at no cost to research laboratories with an interest in discovering new TB drugs. The TAACF is a consortium of contracts managed and funded by the National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, MD) as a resource to support preclinical drug discovery and development. The core of the TAACF is the Southern Research Institute, Birmingham, AL, which supports compound acquisition, storage, medicinal chemistry, and high throughput assays. Other collaborating groups provide biological data on antimycobacterial activity and cytotoxicity, preliminary in vivo toxicity, oral bioavailability and efficacy in animal models, specialty testing (such as activity against non-replicating persistent bacteria), and assistance in technology transfer for developing comprehensive promotional packages and facilitating partnerships with pharmaceutical companies for drug development. The TAACF program and recent progress that has been publicly disclosed by suppliers is reviewed. There are many aspects promising of the program that will not be discussed due to confidentially.

Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening.

Epithelial polarity and tight junction formation limit the ability of adenovirus, retrovirus and adeno-associated virus (AAV) to deliver and express virally encoded genes. Using an extended half-life luciferase assay and high-throughput luminometry, we screened 23 000 compounds and natural product extracts as potentiators to overcome this barrier. Seven strong activators were discovered (up to several hundred fold above control) and two of these exhibited spectrum of activity in multiple cell types (HeLa (human cervical carcinoma), cystic fibrosis bronchial epithelial (human bronchial), HT29 (human colonic carcinoma), Calu3 (airway serous glandular)). Enhanced transduction by unrelated gene transfer vectors (adenovirus, lentivirus, AAV, liposomal) was also observed. These results establish a strategy for identifying compounds that improve viral gene transfer to resistant cell types, and provide new tools for examining epithelial defense against viral infection. The compounds should have broad usefulness in experimental therapies for cancer and genetic diseases.

Studies on (beta,1-->5) and (beta,1-->6) linked octyl Gal(f) disaccharides as substrates for mycobacterial galactosyltransferase activity.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(beta,1-->5)Galf and octyl Galf(beta,1-->6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[14C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.

Studies on alpha(1-->5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity.

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.

Synthesis of disaccharides related to the mycobacterial arabinogalactan.

Several novel glycofuranoses disaccharides related to mycobacterial cell wall polysaccharides were synthesized regio- and stereoselectively using 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranosyl trichloroacetimidate as a glycosyl donor.

Antimycobacterial activity of 1-deaza-7,8-dihydropteridine derivatives against Mycobacterium tuberculosis and Mycobacterium avium complex in vitro.

Twenty-five 1-deaza-7,8-dihydropteridine derivatives were screened for antimycobacterial activity against Mycobacterium tuberculosis strain H37Ra and three Mycobacterium avium clinical isolates (serovar 1, 4 or 6). Antibacterial activity was determined with a colorimetric microdilution broth assay. Seventeen of the compounds inhibited growth in the range >1.28 to

Ethambutol-sugar hybrids as potential inhibitors of mycobacterial cell-wall biosynthesis.

Ethambutol is an established front-line agent for the treatment of tuberculosis, and is also active against Mycobacterium avium infection. However, this agent exhibits toxicity, and is considered to have low potency. The action of ethambutol on the mycobacterial cell wall, particularly the arabinan, and comparison of the structure of ethambutol with several of the cell-wall saccharides, suggested that ethambutol-saccharide hybrids might lead to agents with a more selective mechanism of action. To this end, eight ethambutol-saccharide hybrids were synthesized and screened against M. tuberculosis and several clinical isolates of M. avium.

Studies on beta-D-Gal(f)-(1-->4)-alpha-L-Rha(p) octyl analogues as substrates for mycobacterial galactosyl transferase activity.

The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[14C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three beta-D-gal(f)(1-->4)-alpha-L-rham(p) octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their Km and Vmax values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).

Screening of compounds for antimicrosporidial activity in vitro.

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.

Homologated aza analogs of arabinose as antimycobacterial agents.

A series of hydrolytically-stable aza analogs of arabinofuranose was prepared and evaluated against Mycobacterium tuberculosis and M. avium. The compounds were designed to mimic the putative arabinose donor involved in biogenesis of the essential cell wall polysaccharide, arabinogalactan. Though most compounds displayed little activity in cell culture, one compound showed significant activity in infected macrophage models.

Studies on analogues of classical antifolates bearing the naphthoyl group in place of benzoyl in the side chain.

Analogues of classical antifolates with the 4-aminobenzoyl group replaced by 4-amino-1-naphthoyl were synthesized for study after molecular modeling indicated ample spatial accommodation for the naphthalene ring and even larger groups in models based on reported X-ray crystallographic data describing the binding of methotrexate to human dihydrofolate reductase (DHFR). The side-chain precursors, N-(4-amino- and 4-(methylamino)-1-naphthoyl)-L-glutamic acid diethyl esters, were synthesized, and the 2,4-diamino-substituted heterocyclic groups were attached using several methods. Target compounds included naphthoyl analogues of aminopterin (AMT), methotrexate (MTX), 5-deazaAMT, 5-deazaMTX, 5-methyl-5-deazaAMT, 5-methyl-5-deazaMTX, and 5,8-dideazaAMT. A 5,6,7,8-tetrahydronaphthoyl analogue of 5-deazaAMT was also prepared. None of the naphthoyl analogues showed loss in binding to DHFR compared with the corresponding antifolate bearing the benzoyl group, thus confirming the anticipated bulk tolerance. Only the 5,6,7,8-tetrahydronaphthoyl analogue displayed reduced antifolate effects. Substrate activity toward folylpolyglutamate synthetase was, however, severely compromised. The naphthoyl compounds were transported into L1210 cells 3-6 times more readily than MTX, and despite apparently low levels of intracellular polyglutamylation, each compound was found to be significantly more potent than MTX in inhibiting tumor cell growth in vitro in three lines (L1210, HL60, and S180). The MTX, 5-methyl-5-deazaAMT, and 5-methyl-5-deazaMTX analogues were evaluated in vivo alongside MTX against E0771 mammary adenocarcinoma in mice. All three proved more effective than MTX in retarding the tumor growth. The naphthoyl analogue of 5-deazaAMT strongly inhibited DHFR from Pneumocystis carinii, Toxoplasma gondii, and rat liver giving IC50 (pM) values of 0.53, 2.1, and 1.6 respectively, but this compound did not inhibit in vitro growth of T. gondii, thus indicating lack of transport.

Progress toward the synthesis of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages.

The synthesis of building blocks for the preparation of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages is described. Coupling conditions for the conversion of several of these monomers to dimers are also described.