David Beer

Ten years of dengue drug discovery: progress and prospects.

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITDs missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy.

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

Background The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. Methodology In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. Conclusion The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

The Identification of Indacaterol as an Ultralong-Acting Inhaled β2-Adrenoceptor Agonist

Following a lipophilicity-based hypothesis, an 8-hydroxyquinolinone 2-aminoindan derived series of beta(2)-adrenoceptor agonists have been prepared and evaluated for their potential as inhaled ultralong-acting bronchodilators. Determination of their activities at the human beta(2)-adrenoceptor receptor showed symmetrical substitution of the 2-aminoindan moiety at the 5- and 6-positions delivered the targeted intermediate potency and intrinsic-efficacy profiles relative to a series of clinical reference beta(2)-adrenoceptor agonists. Further assessment with an in vitro superfused electrically stimulated guinea-pig tracheal-strip assay established the onset and duration of action time courses, which could be rationalized by considering the lipophilicity, potency, and intrinsic efficacy of the compounds. From these studies the 5,6-diethylindan analogue indacaterol 1c was shown to possess a unique profile of combining a rapid onset of action with a long duration of action. Further in vivo profiling of 1c supported the long duration of action and a wide therapeutic index following administration to the lung, which led to the compound being selected as a development candidate.

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assays utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.

Indolcarboxamide Is a Preclinical Candidate for Treating Multidrug-Resistant Tuberculosis

The small-molecule indolcarboxamide is a potential drug candidate for treating multidrug-resistant tuberculosis. Combating the Scourge of TB Tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis (Mtb) continues to be an epidemic in many parts of the world. Resistance to multiple drugs and the emergence of the HIV epidemic have created new challenges in TB treatment. Drugs with new mechanisms of action and improved safety profiles are urgently needed to manage TB. To achieve this goal, Rao et al. screened a chemical library of nearly 2 million compounds for inhibitors of mycobacterial growth. Using phenotypic high-throughput screening, they identified a group of molecules called indolcarboxamides as a new class of antitubercular bactericidal agents. Several indolcarboxamide analogs were evaluated to optimize their activity against Mtb and improve their properties. Two lead candidates, NITD-304 and NITD-349, with promising in vivo pharmacokinetic profiles showed potent activity against both drug-sensitive and multidrug-resistant Mtb clinical isolates. Investigating the mechanism of action, the authors found that the molecular target of the indolcarboxamides was MmpL3, a protein that is essential for mycobacterial cell wall biosynthesis and growth. NITD-304 and NITD-349 were efficacious in treating Mtb infections in mouse models of acute and chronic TB with a favorable safety margin. NITD-304 and NITD-349 are promising new drug candidates for treating TB with the potential to help fill the gap in the global TB drug discovery portfolio. New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.

Lipiarmycin targets RNA polymerase and has good activity against multidrug-resistant strains of Mycobacterium tuberculosis

OBJECTIVES The aim of this study was to determine the in vitro activity of lipiarmycin against drug-resistant strains of Mycobacterium tuberculosis (MTB) and to establish the resistance mechanism of MTB against lipiarmycin using genetic approaches. METHODS MIC values were measured against a panel of drug-resistant strains of MTB using the broth microdilution method. Spontaneous lipiarmycin-resistant mutants of MTB were tested for cross-resistance to standard anti-TB drugs, and their rpoB and rpoC genes were sequenced to identify mutations. RESULTS Lipiarmycin exhibited excellent inhibitory activity against multidrug-resistant strains of MTB with MIC values of <0.1 mg/L. Sequence analysis of the rpoB and rpoC genes from spontaneous lipiarmycin-resistant mutants of MTB revealed that missense mutations in these genes caused resistance to lipiarmycin. Although both lipiarmycin and rifampicin are known to inhibit the bacterial RNA polymerase, the sites of mutation in the rpoB gene were found to be different in MTB strains resistant to these inhibitors. Whereas all six rifampicin-resistant MTB strains tested had mutation in the 81 bp hotspot region of the rpoB gene spanning codons 507-533, 16 of 18 lipiarmycin-resistant strains exhibited mutation between codons 977 and 1150. The remaining two lipiarmycin-resistant strains had mutation in the rpoC gene. CONCLUSIONS Lipiarmycin has excellent bactericidal activity against MTB and lacks cross-resistance to standard anti-TB drugs. Furthermore, rifampicin-resistant strains remained fully susceptible to lipiarmycin, and none of the lipiarmycin-resistant MTB strains became resistant to rifampicin, highlighting the lack of cross-resistance.

Direct inhibitors of InhA are active against Mycobacterium tuberculosis

4-Hydroxy-2-pyridones, direct inhibitors of the mycobacterial protein InhA, are active against multidrug-resistant Mycobacterium tuberculosis. “Rediscovering” InhA for Treating TB Isoniazid, a key component of the drug combination currently used to treat tuberculosis, inhibits the Mycobacterium tuberculosis InhA enzyme. Unfortunately, isoniazid has been rendered increasingly obsolete with the spread of multidrug-resistant tuberculosis (MDR-TB). Through phenotypic screening and subsequent target identification, Manjunatha et al. discovered 4-hydroxy-2-pyridones, a new class of InhA inhibitors. Their direct mode of binding to InhA circumvents the main mechanisms of isoniazid resistance, and these compounds showed activity against a number of MDR-TB clinical isolates. Preliminary medicinal chemistry efforts yielded a lead compound NITD-916 that displayed potent oral activity in mouse models of tuberculosis. The structural data presented in this new study provide a path for further optimization of 4-hydroxy-2-pyridones through rational design. New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)–dependent manner and blocked the enoyl substrate–binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB.

Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents

ABSTRACT Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10−7 in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents.

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-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBTPPi, 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 IC50 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-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.