A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.

Design, synthesis and in vitro evaluation of novel SARS-CoV-2 3CLpro covalent inhibitors.

Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CLpro (main coronaviruses cysteine-protease) has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CLpro non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CLpro. We report herein our efforts in the design and synthesis of submicromolar covalent inhibitors when the enzymatic activity of the viral protease was used as a screening platform.

Discovery of covalent prolyl oligopeptidase boronic ester inhibitors.

Over the past decade, many drug discovery endeavors have been invested in targeting the serine proteases prolyl oligopeptidase (POP) for the treatment of Alzheimer's and Parkinson's disease and, more recently, epithelial cancers. Our research group has focused on the discovery of reversible covalent inhibitors, namely nitriles, to target the catalytic serine residue in this enzyme. While there have been many inhibitors discovered containing a nitrile to covalently bind to the catalytic serine, we have been investigating others, particularly boronic acids and boronic esters, the latter of which have been largely unexplored as covalent warheads. Herein we report a series of computationally-designed POP boronic ester pro-drug inhibitors exhibiting nanomolar-potencies in vitro as their active boronic acid species. These easily-accessible (1-2 step syntheses) compounds could facilitate future biochemical and biological studies of this enzyme's role in neurodegenerative diseases and cancer progression.

2'-fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity.

The synthesis of oligonucleotides containing 2'-deoxy-2'-fluoro-4'-thioarabinonucleotides is described. 2'-Deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4'S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4'S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4'-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2'-deoxy-2'-fluoro-4'-thioarabinouridine (4'S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2'F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.

Integrated Synthetic, Biophysical, and Computational Investigations of Covalent Inhibitors of Prolyl Oligopeptidase and Fibroblast Activation Protein α.

Over the past decade, there has been increasing interest in covalent inhibition as a drug design strategy. Our own interest in the development of prolyl oligopeptidase (POP) and fibroblast activation protein α (FAP) covalent inhibitors has led us to question whether these two serine proteases were equal in terms of their reactivity toward electrophilic warheads. To streamline such investigations, we exploited both computational and experimental methods to investigate the influence of different reactive groups on both potency and binding kinetics using both our own series of POP inhibitors and others' discovered hits. A direct correlation between inhibitor reactivity and residence time was demonstrated through quantum mechanics methods and further supported by experimental studies. This computational method was also successfully applied to FAP, as an overview of known FAP inhibitors confirmed our computational predictions that more reactive warheads (e.g., boronic acids) must be employed to inhibit FAP than for POP.

Synthesis and biophysical characterization of oligonucleotides containing a 4'-selenonucleotide.

The first synthesis of oligonucleotides containing 4'-selenium-modified ribonucleotides (4'-Se-rN) is described. Four sequences containing 4'-Se-rT were successfully synthesized and compared with DNA and RNA oligonucleotides containing a dT, rT, or LNA insert in place of the 4'-Se-rT. The 4'-Se-rT behaved more like rT than dT in its effects on binding affinity, despite the DNA-like structure previously observed for the nucleoside, suggesting that a conformational switch occurs upon incorporation into an oligonucleotide. Incorporation of 4'-Se-rT into A-RNA and hybrid duplexes led to increased binding affinity, while incorporation into B-DNA destabilized the duplex to the same extent as an rT nucleotide.

Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT.

A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT. We found that DNA primers that contain North puckered-nucleotides at the 3'-terminus can also affect the translocation status of the RT/template/primer complex, which provides an underlying mechanism to avoid being excised. Together, these results point to a correlation between the sugar conformation of the 3'-terminal nucleotide, the precise position of HIV-1 RT on its nucleic acid substrate, and, in turn, its catalytic function. Nucleotide sugar conformation is therefore an important parameter in defining the susceptibility to RT-catalyzed phosphorolytic excision.

Base-cleavable microarrays for the characterization of DNA and RNA oligonucleotides synthesized in situ by photolithography.

Assessing synthesis efficiency, errors, failed deprotections, and chemical and enzymatic degradation of oligonucleotides on microarrays is essential for improving existing in situ synthesis methods, and for the development of new chemistries. We describe the use of LC-MS to analyse DNA and RNA oligonucleotides deprotected and cleaved under basic conditions from microarrays fabricated using light-directed in situ chemistry. The data yield essential information on array quality and sequence identity.

Phenylpyrrolocytosine as an unobtrusive base modification for monitoring activity and cellular trafficking of siRNA.

6-Phenylpyrrolocytosine (PhpC) is a cytosine mimic with excellent base-pairing fidelity, thermal stability, and high fluorescence. In this work, PhpC-containing small interfering RNAs (siRNAs) are shown to possess thermal stability and gene silencing activity that is virtually identical to that of natural siRNA. The emissive properties of PhpC allow the cellular trafficking of PhpC-containing siRNAs to be monitored by fluorescence microscopy. Accumulation in the cytoplasm of HeLa cells was observed using real time imaging. These findings demonstrate that PhpC-modified siRNAs retain the properties of natural siRNAs while allowing for fluorescence-based detection and monitoring, providing an ideal system for probing siRNA uptake and trafficking.

RNA containing pyrrolocytidine base analogs: increased binding affinity and fluorescence that responds to hybridization.

6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. These analogs produce increased binding affinity to both RNA and DNA targets when incorporated into oligoribonucleotides. The fluorescence observed for the single-stranded oligomers is quenched upon duplex formation with either RNA or DNA targets. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside.

RNA containing pyrrolocytidine base analogs: good binding affinity and fluorescence that responds to hybridization.

6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. When incorporated into RNA, these analogs enhance binding affinity towards RNA and DNA targets with a concomitant change in their fluorescence upon duplex formation. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside. The fluorescence response of these structurally conservative, well-tolerated fluorescent nucleosides may be exploited as probes in the study of nucleic acid processing enzymes.