High-affinity fluorescent ligands for the 5-HT(3) receptor.

The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT(3) receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT(3)AR.

Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability.

Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (10, 19, 20 and 26) with markedly improved metabolic stability. Compared to PF74, analog 20 exhibited similar submicromolar potency, and much longer (51-fold) half-life in human liver microsomes (HLMs). Molecular docking corroborated that 20 binds to the PF74 binding site, and revealed distinct binding interactions conferred by the benzamide moiety. Collectively, our data support compound 20 as a promising antiviral lead.

Design, Synthesis and Characterization of HIV-1 CA-Targeting Small Molecules: Conformational Restriction of PF74.

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 µM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.

Novel HIV-1 capsid-targeting small molecules of the PF74 binding site.

The PF74 binding site in HIV-1 capsid protein (CA) is a compelling antiviral drug target. Although PF74 confers mechanistically distinct antiviral phenotypes by competing against host factors for CA binding, it suffers from prohibitively low metabolic stability. Therefore, there has been increasing interest in designing novel sub-chemotypes of PF74 with similar binding mode and improved metabolic stability. We report herein our efforts to explore the inter-domain interacting indole moiety for designing novel CA-targeting small molecules. Our design includes simple substitution on the indole ring, and more importantly, novel sub-chemotypes with the indole moiety replaced with a few less electron-rich rings. All 56 novel analogs were synthesized and evaluated for antiviral activity, cytotoxicity, and impact on CA hexamer stability. Selected analogs were tested for metabolic stability in liver microsomes. Molecular modeling was performed to verify compound binding to the PF74 site. In the end, 5-hydroxyindole analogs (8,9 and 12) showed improved potency (up to 20-fold) over PF74. Of the novel sub-chemotypes, α- and ß-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.

Toward Structurally Novel and Metabolically Stable HIV-1 Capsid-Targeting Small Molecules.

HIV-1 capsid protein (CA) plays an important role in many steps of viral replication and represents an appealing antiviral target. Several CA-targeting small molecules of various chemotypes have been studied, but the peptidomimetic PF74 has drawn particular interest due to its potent antiviral activity, well-characterized binding mode, and unique mechanism of action. Importantly, PF74 competes against important host factors for binding, conferring highly desirable antiviral phenotypes. However, further development of PF74 is hindered by its prohibitively poor metabolic stability, which necessitates the search for structurally novel and metabolically stable chemotypes. We have conducted a pharmacophore-based shape similarity search for compounds mimicking PF74. We report herein the analog synthesis and structure-activity relationship (SAR) of two hits from the search, and a third hit designed via molecular hybridization. All analogs were characterized for their effect on CA hexamer stability, antiviral activity, and cytotoxicity. These assays identified three active compounds that moderately stabilize CA hexamer and inhibit HIV-1. The most potent analog (10) inhibited HIV-1 comparably to PF74 but demonstrated drastically improved metabolic stability in liver microsomes (31 min vs. 0.7 min t1/2). Collectively, the current studies identified a structurally novel and metabolically stable PF74-like chemotype for targeting HIV-1 CA.

Chemical profiling of HIV-1 capsid-targeting antiviral PF74.

The capsid protein (CA) of HIV-1 plays essential roles in multiple steps of the viral replication cycle by assembling into functional capsid core, controlling the kinetics of uncoating and nuclear entry, and interacting with various host factors. Targeting CA represents an attractive yet underexplored antiviral approach. Of all known CA-targeting small molecule chemotypes, the peptidomimetic PF74 is particularly interesting because it binds to the same pocket used by a few important host factors, resulting in highly desirable antiviral phenotypes. However, further development of PF74 entails understanding its pharmacophore and mitigating its poor metabolic stability. We report herein the design, synthesis, and evaluation of a large number of PF74 analogs aiming to provide a comprehensive chemical profiling of PF74 and advance the understanding on its detailed binding mechanism and pharmacophore. The analogs, containing structural variations mainly in the aniline domain and/or the indole domain, were assayed for their effect on stability of CA hexamers, antiviral activity, and cytotoxicity. Selected analogs were also tested for metabolic stability in liver microsomes, alone or in the presence of a CYP3A inhibitor. Collectively, our studies identified important pharmacophore elements and revealed additional binding features of PF74, which could aid in future design of improved ligands to better probe the molecular basis of CA-host factor interactions, design strategies to disrupt them, and ultimately identify viable CA-targeting antiviral leads.

Mapping the Orthosteric Binding Site of the Human 5-HT3 Receptor Using Photo-cross-linking Antagonists.

The serotonin-gated 5-HT3 receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT3 receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [3H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT3 receptor. Detailed analysis by protein-MS/MS identified a residue (Met-228) near the tip of binding loop C as the covalent modification site.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c.

Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing at the C5 position that led to drastically improved RNase H inhibition and significant antiviral activity. Structure-activity relationship (SAR) concerning primarily the length and flexibility of the two wings revealed important structural features that dictate the potency and selectivity of RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry data also revealed that the RNase H biochemical inhibition largely correlated the antiviral activity.

5'-Silylated 3'-1,2,3-triazolyl Thymidine Analogues as Inhibitors of West Nile Virus and Dengue Virus.

West Nile virus (WNV) and Dengue virus (DENV) are important human pathogens for which there are presently no vaccine or specific antivirals. We report herein a 5'-silylated nucleoside scaffold derived from 3'-azidothymidine (AZT) consistently and selectively inhibiting WNV and DENV at low micromolar concentrations. Further synthesis of various triazole bioisosteres demonstrated clear structure-activity relationships (SARs) in which the antiviral activity against WNV and DENV hinges largely on both the 5'-silyl group and the substituent of 3'-triazole or its bioisosteres. Particularly interesting is the 5' silyl group which turns on the antiviral activity against WNV and DENV while abrogating the previously reported antiviral potency against human immunodeficiency virus (HIV-1). The antiviral activity was confirmed through a plaque assay where viral titer reduction was observed in the presence of selected compounds. Molecular modeling and competitive S-adenosyl-l-methionine (SAM) binding assay suggest that these compounds likely confer antiviral activity via binding to methyltransferase (MTase).

Design, synthesis, biochemical, and antiviral evaluations of C6 benzyl and C6 biarylmethyl substituted 2-hydroxylisoquinoline-1,3-diones: dual inhibition against HIV reverse transcriptase-associated RNase H and polymerase with antiviral activities.

Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety. The synthesis involved a recently reported metal-free direct benzylation between tosylhydrazone and boronic acid, which allowed the generation of structural diversity for the hydrophobic aromatic region. Biochemical studies showed that the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes, consistently inhibited HIV RT-associated RNase H and polymerase with IC50s in low to submicromolar range. The observed dual inhibitory activity remained uncompromised against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these same compounds inhibited the polymerase, but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV) RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially reduced level of inhibition against HIV integrase. Molecular docking corroborates favorable binding of these analogues to the active site of HIV RNase H. Finally, a number of these analogues also demonstrated antiviral activity at low micromolar concentrations.

Clicking 3'-azidothymidine into novel potent inhibitors of human immunodeficiency virus.

3'-Azidothymidine (AZT) was the first approved antiviral for the treatment of human immunodeficiency virus (HIV). Reported efforts in clicking the 3'-azido group of AZT have not yielded 1,2,3-triazoles active against HIV or any other viruses. We report herein the first AZT-derived 1,2,3-triazoles with submicromolar potencies against HIV-1. The observed antiviral activities from the cytopathic effect (CPE) based assay were confirmed through a single replication cycle assay. Structure-activity-relationship (SAR) studies revealed two structural features key to antiviral activity: a bulky aromatic ring and the 1,5-substitution pattern on the triazole. Biochemical analysis of the corresponding triphosphates showed lower ATP-mediated nucleotide excision efficiency compared to AZT, which along with molecular modeling suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This mechanism is corroborated with the observed reduction of fold resistance of the triazole analogue to an AZT-resistant HIV variant (9-fold compared to 56-fold with AZT).

5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC50 = 0.87 µM). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.

Toward biophysical probes for the 5-HT3 receptor: structure-activity relationship study of granisetron derivatives.

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.