I Indian Journal of Exprimental Biology Vol. 52, April 2014, pp. 375-382 Pharmacophore based approach to design inhibitors in Crustaceans: An insight into the molt inhibition response to the receptor guanylyl cyclase Sajal Shrivastava & S Adline Princy* Quorum Sensing Laboratory, SASTRA’s Hub for Research and Innovation, SASTRA University, Thanjavur 613 401, India Received 13 May 2013; revised 8 January 2014 The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock. Keywords: Ecdysteroid, Growth enhancers, HipHop, Molt inhibiting hormone, Pharmacophore In crustaceans, molting, growth, and development is in the medullar terminalis of X-organ and then governed by ecdysteroids synthesized by paired transported to the sinus gland8-12. Various endocrine glands, Y-organs that are located in the quantification studies have revealed that the quantity anterior cephalothorax1-4. Crustacean hyperglycemic or the number of receptors on the Y-organ remains hormone (CHH) family neuropeptides are potential constant and during intermolt period, the synthesis modulators of various regulatory processes in the of ecdysteroids favorably inactivated by MIH13-15. crustacean physiological system: CHH stimulate Contemporary reports from radioreceptor binding during stress conditions such as hypoxia, hyper assays also intend a relation between the rate of or hypothermia, molt inhibiting hormone (MIH) ecdysteroid production and the receptiveness of the inhibits the synthesis and/or secretion of ecdysteroids Y-organ to MIH, but the MIH receptor has not been from Y-organ and vitellogenin inhibitory peptide fully characterized for any species16-20. Inhibition (VIH) inhibits the reproduction. The CHH family of facultative synthesis of ecdysteroid by MIH is neuropeptides reveal pleotropic properties, i.e. the facilitated by activation of the specific transcription neuropeptides having more than two diverse factor that impedes phantom expression21. It has been biological activities have been described5-7. The most reported that MIH predominantly binds to guanylyl extensively acknowledged paradigm proposes that cyclase (GC-II) and/or G protein coupled receptor synthesis of ecdysteroids, polyhydroxylated C-27 on the Y-organ cells and suppresses the ecdysteroid steroid derived from cholesterol is negatively biosynthesis by a cAMP-dependent activation of regulated by molt inhibiting hormone synthesized nitric oxide synthase (NOS) and NO-dependent guanylyl cyclase (GC-I), both of which are articulated —————— 22-27 * in YOs . The existence of MIH, one of the most Correspondent author critical neuropeptide in the family of CHH was Telephone: +91 4362 264101 Fax: +91 4362 264120 suggested by the fact that eye-stalk ablation leads to E-mail: [email protected] prompt upsurge in ecdysteroid titer in hemolymph 376 INDIAN J EXP BIOL, APRIL 2014 and hence, persuades precocious molting28. However, and functional specificity and the homology model as CHH, gonad inhibiting hormone (GIH) and of CHH evidently indicates the absence of the mandibular organ inhibiting hormone (MOIH) are N-terminal α-helix and C-terminal tail. also synthesized in X-organ and secreted from sinus Pharmacophore based approach—To date, the 3D gland, eye-stalk ablation cause imbalances in other structure of receptor guanylyl cyclase has not been physiological processes29,30. obtained by the experiment and the homology Structure of molt inhibiting hormone (MIH)—MIH module packages were unsuccessful to build the has been sequestered and characterized from decapod initial model of rGC due to very little sequence and crustaceans of several taxa and it was apparent that structural resemblances in the database. Additional the size of mature MIH ranges from 8-11 kDa and structural information, notably of the receptor are 72-78 amino acid residues in length. Reports guanylyl cyclase in either free or MIH-bound state, strongly indicate the presence of six highly conserved will be very helpful for the optimization of inhibitors, cysteine residues in all CHH family neuropeptides such as to increase frequency of molting to achieve that are responsible for the formation of the better growth in crustaceans. three disulfide bridges to stabilize their structure31. In absence of a three-dimensional structure of Where pertinent data exist, most MIH comprise receptor guanylyl cyclase and data from crystal a non-amidated C-terminus whereas CHH have an structure and binding site of molt inhibiting hormone, amidated C-terminus substantial for conferring pharmacophore-based design of competitive hyperglycemic activity32. The solution structure of inhibitors is one of the ubiquitous approaches for MIH from the kuruma prawn, Marsupenaeus drug discovery and lead optimization. The discovery japonicus reveals the existence of five α-helices of the three dimensional structure of molt inhibiting located within the N-terminal motif in the hormone by X-ray crystallography initiated the way MIH structure and was considered a part of the for design and synthesis of competitive inhibitors. determinants of the functional specificity33-35. MIH as a therapeutically relevant drug target with Mutation analysis has validated that the N-terminal undetermined active site geometries, HipHop based N13 and C-terminal S71 and I72 residues are pharmacophore modeling provides an effective especially significant for conferring molt inhibiting mechanism for virtual screening. In recent years, activity (Fig. 1) and these two motifs were close to the pharmacophore-based approach has become very each other in the 3-dimensional spatial arrangement36. commanding for the discovery of novel lead Since the binding of MIH with receptors in the compounds and for lead optimization. In the Y-organ suppresses the growth and development in present study, molecular recognition techniques have crustaceans, a transient interference with binding of been employed including HipHop to screen potential MIH to its receptor can promote the growth37-39. drug candidates in silico and manually modified to Most of the CHH family peptides exhibit biological produce novel drug like compounds. Pharmacophore Fig.1—Multiple sequence alignment of mature Molt inhibiting hormones from representative crustacean species [The residues are numbered from the first residue of mature peptide and the amino acids critical for the activities of MIH are indicated by asterisks (*). The sources for MIH sequences considered for multiple sequence alignment are as follows: CAR- Carcinus maenas (GenBank accession number: CAA53591); CAL- Callinectes sapidus (GenBank accession number: AAA69029); CAN- Cancer pagurus (GenBank accession number: CAC39425); CHAR- Charybdis feriatus (GenBank accession number: AAC64785); ORC- Orconectes limosus (UniProtKB/Swiss-Prot accession number: P83636); PRO- Procambarus clarkii (UniProtKB/Swiss-Prot accession: P55848); PEN- Penaeus japonicus (UniProtKB/Swiss-Prot accession number: P55847); MET- Metapenaeus ensis (GenBank accession number: AAC27452); LIT- Litopenaeus vannamei (GenBank accession number: ABD73292); TRA- Trachypenaeus curvirostris (GenBank accession number: AF312978)] SHRIVASTAVA & PRINCY: MOLT INHIBITING RESPONSE TO GUANYLYL CYCLASE IN CRUSTACEANS 377 modeling has been adopted to rapidly identify new Structural analysis and selection of training sets— potential drugs to overcome dawdling growth by The co-ordinates and the spatial arrangement of interfering with the interaction of MIH with its residues of MIH were obtained from refined X-ray receptor. Pharmacophore models for the MIH- structure of Marsupenaeus japonicus (MJ-MIH) antagonists have been generated using HipHop which was retrieved from the Protein Data Bank algorithm entailing identification and overlay (accession code: 1J0T). The most critical task in the common features. These models are anticipated drug discovery process is developing an appropriate to provide a rational hypothetical picture of the model to predict