Discovery of novel drug targets and their functions using phenotypic screening of natural products

Natural products are valuable resources that provide a variety of bioactive compounds and natural pharmacophores in modern drug discovery. Discovery of biologically active natural products and unraveling their target proteins to understand their mode of action have always been critical hurdles for their development into clinical drugs. For effective discovery and development of bioactive natural products into novel therapeutic drugs, comprehensive screening and identification of target proteins are indispensable. In this review, a systematic approach to understanding the mode of action of natural products isolated using phenotypic screening involving chemical proteomics-based target identification is introduced. This review highlights three natural products recently discovered via phenotypic screening, namely glucopiericidin A, ecumicin, and terpestacin, as representative case studies to revisit the pivotal role of natural products as powerful tools in discovering the novel functions and druggability of targets in biological systems and pathological diseases of interest.     

Development of small-molecule P-gp inhibitors of the N-benzyl 1,4-dihydropyridine type: Novel aspects in SAR and bioanalytical evaluation of multidrug resistance (MDR) reversal properties

Novel series of N-benzyl 1,4-dihydropyridines have been prepared by facile syntheses. All relevant substituents of the molecular scaffold have been varied. The resulting compounds were biologically evaluated as P-glycoprotein (P-gp) inhibitors. Substitutions of the N-benzyl residue favour biological activity beside respective 3-ester functions. Most active compounds were further evaluated as multidrug resistance (MDR) modulators to restore the cytotoxic properties of varying daunorubicin applications.     

Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments.

Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.     

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti.

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.     

Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents

We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure–activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS–PAGE gels. This stepwise, ‘tag-free’ approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.     

4-Substituted quinazoline derivatives as novel EphA2 receptor tyrosine kinase inhibitors

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.     

Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases

Activity-based probes (ABPs) have found increasing use in functional proteomics studies. Recently, ABPs that can be employed in combination with click chemistry gained particular attention due to their flexible application in vitro and in vivo. Moreover, there is a continuous need for new ABPs that target small subsets of enzymes. We here report novel clickable ABPs based on the 4-chloro-isocoumarin (IC) electrophile, a mechanism-based inhibitor scaffold that covalently binds serine proteases. We describe the synthesis of a small library of IC ABPs containing an alkyne function and a set of diverse selectivity elements. The different substituents on the IC structure determine which proteases are bound, showing good correlation with the preferred substrate preferences. The IC ABPs can detect their target proteases in a proteome background in a sensitive manner (down to 0.007% of total protein). Furthermore, we show activity-dependent and selective labeling of endogenous proteases in a tissue proteome. These ICs therefore represent a valuable extension to already existing ABPs for serine proteases and may be instrumental in future elucidation of serine protease functions.     

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4'-diacetyldiphenylurea bis(guanylhydrazone) (DDUG)

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.     

Reactive oxygen-dependent production of novel photochemotherapeutic agents.

The reactive nature of species derived from oxygen, such as singlet oxygen and hydrogen peroxide, has been exploited in the clinical setting for targeting bacteria, viruses, and tumor cells by photodynamic excitation of a variety of chromophores. This modality, termed photodynamic therapy (PDT), is currently being used to treat some forms of cancer. However, the applicability of conventional PDT is limited due to the absolute dependence on simultaneous exposure of the target to the photoactive compound and light. In 1990, we demonstrated that the need for simultaneous exposure of the biological target to light and photosensitizer could be circumvented by prior exposure (activation) of the sensitizer molecule to light and its subsequent use as any other anti-cancer or anti-viral drug. By dint of the nature of the protocol, this process was termed preactivation. Since then, the generation of biologically active molecules in vitro by preactivation has been validated using a variety of chromophores, such as merocyanine 540, Photofrin II, and naphthalimide. Here we briefly review the role of reactive oxygen species in the photodynamic effect, and provide an explanation for the mechanism of preactivation. We propose that photo-oxidation not only provides a novel means for the generation of biologically active molecules, but could also explain, at least in part the mechanism of conventional PDT. It is likely that the light-dependent breakdown of the chromophore to generate novel active compounds, in addition to reactive oxygen species, also contributes to the photodynamic damage observed on simultaneous exposure of the chromophore and target tissue to light during PDT.-Pervaiz, S. Reactive oxygen-dependent production of novel photochemotherapeutic agents.     

Synthesis,spectral and antimicrobial evaluation of some novel 1-methyl-3-alkyl-2,6-diphenylpiperidin-4-one oxime carbonates

Synthesis of some novel biologically active piperidin-4-one oxime carbonates from 1-methyl-3alkyl-2,6-diphenylpiperidin-4-one oximes and substituted chloroformates was carried out in the presence of potassium carbonate as base and tetrabutylammonium bromide (TBAB) as catalyst. The newly synthesized compounds were characterized by IR, ¹H, ¹³C NMR and LC–mass spectra. Based on the ¹H NMR analysis, all the compounds were found to adopt normal chair conformation with equatorial orientation of all the substituents. For all the synthesized compounds (5a–5l) antimicrobial activity has been tested against bacterial and fungal strains using Streptomycin and Amphotericin B as standards.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

Synthesis and biological evaluation of novel 2,4-disubstituted quinazoline analogues as GPR119 agonists

GPR119 agonist has emerged as a promising target for the treatment of type 2 diabetes. A series of novel 2,4-disubstituted quinazoline analogues was prepared and evaluated their agonistic activity against human GPR119. The analogues bearing azabicyclic amine substituents (12a, 12c and 12g) exhibited better EC₅₀ values than that of OEA though they appeared to be partial agonists.     

Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: design,synthesis and structure-activity relationship

The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1' hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1' directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1' directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1' directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.     

Probing the active site of reconstituted human placental aromatase [Poster 02]

To gain some insight into the factors that determine active site-ligand binding and to delineate which sites of the androstene nucleus are critical for recognition by the aromatase enzyme, we compared the effect of various substituents located at the biologically important C-2, 6, 11, 16, 17, and 19 positions. Based on two independent measurements, namely optical difference spectra and inhibition of enzyme activity, we report: (a) oxygen functions at positions C-11 do not appear to induce difference spectra although they cause weak inhibition; (b) 2ß- and 16-hydroxyandrostenes induce type-I spectra and weak inhibition; (c) C-6 and C-19-substituted androgens are type-I spectrum inducers and strong inhibitors; (d) the presence of nonspecific binding sites on the aromatase enzyme cannot be ruled out.     

Three Drosophila Hox complex microRNAs do not have major effects on expression of evolutionarily conserved Hox gene targets during embryogenesis

The discovery of microRNAs has resulted in a major expansion of the number of molecules known to be involved in gene regulation. Elucidating the functions of animal microRNAs has posed a significant challenge as their target interactions with messenger RNAs do not adhere to simple rules. Of the thousands of known animal microRNAs, relatively few microRNA:messenger RNA regulatory interactions have been biologically validated in an normal organismal context. Here we present evidence that three microRNAs from the Hox complex in Drosophila (miR-10-5p, miR-10-3p, miR-iab-4-5p) do not have significant effects during embryogenesis on the expression of Hox genes that contain high confidence microRNAs target sites in the 3' untranslated regions of their messenger RNAs. This is significant, in that it suggests that many predicted microRNA-target interactions may not be biologically relevant, or that the outcomes of these interactions may be so subtle that mutants may only show phenotypes in specific contexts, such as in environmental stress conditions, or in combinations with other microRNA mutations.     

Targeting RNA with cysteine-constrained peptides

A combined approach for targeting RNA with novel, biologically active ligands has been developed using a cyclic peptide library and in silico modeling. This approach has successfully identified novel cyclic peptide constructs that can target bTAR RNA. Subsequently, RNA/peptide interactions were effectively modeled using the HADDOCK docking program.     

Synthesis and biological evaluation of some 6-arylamidomorphines as analogues of morphine-6-glucuronide

A series of 6-beta-arylamidomorphines was synthesized and biologically evaluated. Various aryl substituents were introduced into the arylamidomorphines to examine substituent structure-activity relationships. Competition binding assays showed that compounds 10a-h bound to the mu opioid receptor with high affinity (0.2-0.6 nM). Functional assays showed that compounds 10a-h acted as full mu opioid receptor agonists. The ED(50) of compound 10e.HCl as an analgesic was 12.6 mg/kg in the tail flick latency test in the rat.     

New reagents, reactions, and peptidomimetics for drug design.

It has been a major focus in our laboratories to prepare novel reagents and peptidomimetic structures for drug design. We have designed and prepared novel guanidinylation reagents that can be employed in solution or as solid phase reagents. We and others have utilized the reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) for amide bond formation to couple sterically hindered structures. These couplings proceed with remarkably strong resistance to racemization. In the area of peptidomimetics, we have incorporated novel building blocks to create biologically active compounds. These building blocks include thioether and alkylamine bridges, beta-methylated, and beta,beta-dimethylated amino acid residues. These mimetic structures have been incorporated into specific target molecules such as opioids to obtain cyclic peptidomimetics with potent and selective biological activity.