A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3'UTR-U(30) RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT(PPi), which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3'dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC(50) values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3'UTR-C(30) RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.     

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)₂₀]. Chemical modification of the initial “hit” improved the compound potency to an IC₅₀ (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC₅₀ of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC₅₀s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.The family Flaviviridae consists of three genera: Flavivirus, Pestivirus, and Hepacivirus. The genus Flavivirus contains about 73 viruses, many of which are arthropod-borne and pose major public health threats worldwide (15). The four serotypes of dengue virus infect 50 to 100 million people each year, with approximately 500,000 cases developing into life-threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS), leading to about 20,000 deaths. In addition to DENV, West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) also cause significant human diseases. No antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are only available for YFV, JEV, and TBEV (15). Development of antiviral therapy and new vaccines is urgently needed for flaviviruses.The flavivirus genome is a single-stranded RNA of plus-sense polarity. The genomic RNA contains a 5′ untranslated region (UTR), a single open reading frame, and a 3′ UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins. Three structural proteins (Capsid [C], premembrane [prM], and envelope [E]) are components of virus particles. Seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are responsible for viral replication (40), virion assembly (19, 21, 24, 33), and innate immunity antagonism (4, 16, 23, 29, 30). Two viral proteins encode enzymatic activities that have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5′-RNA triphosphatase, and nucleoside triphosphatase (7, 14, 42). The N-terminal part of NS5 is a methyltransferase that methylates the N7 and 2′-O positions of the viral RNA cap structure (13, 18, 37); the C-terminal part of NS5 has an RNA-dependent RNA polymerase (RdRp) activity (1, 39). The RdRp activity is unique to RNA viruses and therefore represents an attractive antiviral target.Two types of inhibitors could be developed to suppress viral polymerases. Type 1 inhibitors are nucleoside/nucleotide analogs that function as RNA or DNA chain terminators; about half of the current antiviral drugs are nucleotide analogs (10). For flaviviruses, a nucleoside analog (7-deaza-2′-C-methyl-adenosine), originally developed for hepatitis C virus (HCV) RdRp, showed anti-DENV activity (32, 38). We recently reported a similar adenosine analog (7-deaza-2′-C-acetylene-adenosine) that potently inhibited DENV both in cell culture and in mice; unfortunately, this compound showed side effects during a 2-week in vivo toxicity study (44). Nevertheless, these studies have proved the concept that nucleoside analogs could potentially be developed for flavivirus therapy. Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the mechanism of action of NNI includes structural alteration of polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or impeding the processivity of polymerase elongation (11). A broad range of chemical classes have been identified as NNI, including inhibitors of HIV (9, 35) and HCV (3, 5, 11, 25).In the present study, we performed high-throughput screening (HTS) to search for NNI of DENV RdRp. The HTS and chemistry synthesis led to the identification of N-sulfonylanthranilic acid derivatives as inhibitors of DENV RdRp. The compounds specifically inhibit DENV RdRp. UV cross-linking experiments mapped the compound binding site to the RdRp domain of DENV NS5. Amino acid Met343, located at the entrance of RNA template tunnel of the DENV RdRp, was cross-linked to the compound. These results, together with biochemistry experiments, suggest that the compound blocks the RdRp activity through binding to the RNA template tunnel of the polymerase.     

Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.     

Biochemical and structural characterization of the putative dihydropteroate synthase ortholog Rv1207 of Mycobacterium tuberculosis

Dihydropteroate synthase (DHPS) is involved in de novo biosynthesis of the essential cofactor folate by catalyzing the condensation of para-aminobenzoic acid (pABA) and 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (H(2)PtPP). Mycobacterium tuberculosis possesses a functional DHPS (MtDHPS, Rv3608c, folP1) and, based on sequence similarities, a putative ortholog (Rv1207, folP2). Here, we demonstrate that Rv1207 shows a low H(2)PtPP substrate affinity and lacks enzymatic DHPS activity. However, we found dapsone, a structural analog of pABA and clinically used DHPS inhibitor, to weakly bind both proteins. To gain insights into the lack of DHPS activity of Rv1207, its three-dimensional structure was determined at 2.64 A. The overall fold of both, MtDHPS (1EYE) and Rv1207, is highly conserved and conforms to a classical triosephosphate isomerase barrel arrangement. The predicted H(2)PtPP-binding pocket of Rv1207 is occupied by a histidine side chain, relative to a leucine residue in MtDHPS, consistent with the low affinity for this substrate and the lack of DHPS activity. We conclude that folP2 does not encode a DHPS and therefore cannot act as bypass for folP1. The metabolic function of Rv1207 remains to be defined.     

Proteochemometrics analysis of substrate interactions with dengue virus NS3 proteases

The prime side specificity of dengue protease substrates was investigated by use of proteochemometrics, a technology for drug target interaction analysis. A set of 48 internally quenched peptides were designed using statistical molecular design (SMD) and assayed with proteases of four subtypes of dengue virus (DEN-1-4) for Michaelis (K(m)) and cleavage rate constants (k(cat)). The data were subjected to proteochemometrics modeling, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. Detailed analysis of the models then showed that considerably differing physico-chemical properties of amino acids contribute independently to the K(m) and k(cat) activities. For k(cat), only P1' and P2' prime side residues were important, while for K(m) all four prime side residues, P1'-P4', were important. The models could be used to identify amino acids for each P' substrate position that are favorable for, respectively, high substrate affinity and cleavage rate.     

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.     

Competitive inhibition of the dengue virus NS3 serine protease by synthetic peptides representing polyprotein cleavage sites

The NS3 serine protease of dengue virus is required for the maturation of the viral polyprotein and consequently represents a promising target for the development of antiviral inhibitors. However, the substrate specificity of this enzyme has been characterized only to a limited extent. In this study, we have investigated product inhibition of the NS3 protease by synthetic peptides derived from the P6-P1 and the P1'-P5' regions of the natural polyprotein substrate. N-terminal cleavage site peptides corresponding to the P6-P1 region of the polyprotein were found to act as competitive inhibitors of the enzyme with K(i) values ranging from 67 to 12 microM. The lowest K(i) value was found for the peptide representing the NS2A/NS2B cleavage site, RTSKKR. Inhibition by this cleavage site sequence was analyzed by using shorter peptides, SKKR, KKR, KR, AGRR, and GKR. With the exception of the peptide AGRR which did not inhibit the protease at a concentration of 1mM, all other peptides displayed K(i) values in the range from 188 to 22 microM. Peptides corresponding to the P1'-P5' region of the polyprotein cleavage sites had no effect on enzymatic activity at a concentration of 1mM. Molecular docking data of peptide inhibitors to a homology-based model of the dengue virus type 2 NS2B(H)-NS3p co-complex indicate that binding of the non-prime site product inhibitors is similar to ground-state binding of the corresponding substrates.     

Inhibition of dengue virus through suppression of host pyrimidine biosynthesis

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC(50)) of 2.4 nM and a 50% cytotoxic concentration (CC(50)) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC(90)s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.