Rational Design of Peptide Ligands Based on Knob−Socket Protein Packing Model Using CD13 as a Prototype Receptor

Structure-based computational peptide design methods have gained significant interest in recent years owing to the availability of structural insights into protein–protein interactions obtained from the crystal structures. The majority of these approaches design new peptide ligands by connecting the crucial amino acid residues from the protein interface and are generally not based on any predicted receptor–ligand interaction. In this work, a peptide design method based on the Knob–Socket model was used to identify the specific ligand residues packed into the receptor interface. This method enables peptide ligands to be designed rationally by predicting amino acid residues that will fit best at the binding site of the receptor protein. In this, specific peptide ligands were designed for the model receptor CD13, overexpression of which has been observed in several cancer types. From the initial library of designed peptides, three potential candidates were selected based on simulated energies in the CD13 binding site using the programs molecular operating environment and AutoDock Vina. In the CD13 enzymatic activity inhibition assay, the three identified peptides exhibited 2.7–7.4 times lower IC50 values (GYPAY, 227 μM; GFPAY, 463 μM; GYPAVYLF, 170 μM) as compared to the known peptide ligand CNGRC (C1–C5) (1260 μM). The apparent binding affinities of the peptides (GYPAY, Ki = 54.0 μM; GFPAY, Ki = 74.3 μM; GYPAVYLF, Ki = 38.8 μM) were 10–20 times higher than that of CNGRC (C1–C5) (Ki = 773 μM). The double reciprocal plots from the steady-state enzyme kinetic assays confirmed the binding of the peptides to the intended active site of CD13. The cell binding and confocal microscopy assays showed that the designed peptides selectively bind to the CD13 on the cell surface. Our study demonstrates the feasibility of a Knob–Socket-based rational design of novel peptide ligands in improving the identification of specific binding versus current more labor-intensive methods

Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets

Basic biology of Trichomonas vaginalis: current explorations and future directions

Trichomonas vaginalis is a member of the Parabasalia, a group of single-celled eukaryotes within the clade Excavata, which also includes parasites of genera such as Giardia and Trypanosoma. The ability to culture the parasite under both aerobic and anaerobic conditions has allowed simple manipulation in the laboratory. Studies of the basic biology of this organism not only provide important information about its unique cellular and metabolic features and contribute to our understanding of the evolution of anaerobic parasites and diversity among eukaryotes, but also contribute to the design of new drugs to cure infections. The publication of the genome sequence in 20071 identified several unique characteristics of the organism, such as the large genome size (∼160 megabases), extensive gene duplication and presence of families of transposable elements. It also facilitated data mining and the generation of ‘omic’ data sets, especially from transcriptomic and proteomic studies

Rational Design of Peptide Ligands Based on Knob−Socket Protein Packing Model Using CD13 as a Prototype Receptor

Structure-based computational peptide design methods have gained significant interest in recent years owing to the availability of structural insights into protein–protein interactions obtained from the crystal structures. The majority of these approaches design new peptide ligands by connecting the crucial amino acid residues from the protein interface and are generally not based on any predicted receptor–ligand interaction. In this work, a peptide design method based on the Knob–Socket model was used to identify the specific ligand residues packed into the receptor interface. This method enables peptide ligands to be designed rationally by predicting amino acid residues that will fit best at the binding site of the receptor protein. In this, specific peptide ligands were designed for the model receptor CD13, overexpression of which has been observed in several cancer types. From the initial library of designed peptides, three potential candidates were selected based on simulated energies in the CD13 binding site using the programs molecular operating environment and AutoDock Vina. In the CD13 enzymatic activity inhibition assay, the three identified peptides exhibited 2.7–7.4 times lower IC50 values (GYPAY, 227 μM; GFPAY, 463 μM; GYPAVYLF, 170 μM) as compared to the known peptide ligand CNGRC (C1–C5) (1260 μM). The apparent binding affinities of the peptides (GYPAY, Ki = 54.0 μM; GFPAY, Ki = 74.3 μM; GYPAVYLF, Ki = 38.8 μM) were 10–20 times higher than that of CNGRC (C1–C5) (Ki = 773 μM). The double reciprocal plots from the steady-state enzyme kinetic assays confirmed the binding of the peptides to the intended active site of CD13. The cell binding and confocal microscopy assays showed that the designed peptides selectively bind to the CD13 on the cell surface. Our study demonstrates the feasibility of a Knob–Socket-based rational design of novel peptide ligands in improving the identification of specific binding versus current more labor-intensive methods

Preliminary Studies of 3,4-Dichloroaniline Amides as Antiparasitic Agents: Structure Activity Analysis of a Compound Library in vitro Against Trichomonas vaginalis

Trichomonas vaginalis, a human-infectious protozoan, can display resistance to treatment by metronidazole. A library of 3,4-dichloroaniline amides based on propanil, an herbicide, has been synthesized and screened to test susceptibility to these analogs. From this preliminary study, the most effective compound 15, inhibits growth of the organism by 66% and 69% on the two strains tested, T1 and G3, respectively

Design and synthesis of b-amino alcohol based b-lactam-isatin chimeras and preliminary analysis of in vitro activity against the protozoal pathogen Trichomonas vaginalis

We have described the synthesis of β-amino alcohol based β-lactam–isatin chimeras along with their in vitro evaluation against T. vaginalis. SAR studies showed dependence of activity on concentration as well as substituent on N-1 of β-lactam and C-5 of isatin with preference for a chloro-substituent for good activity

Synthesis and antiprotozoal activity of mono- and bis-uracil isatin conjugates against the human pathogen Trichomonas vaginalis

A library of mono- and bis-uracil isatin conjugates were synthesized and subjected for the assessment of their in vitro activity against the protozoal pathogen Trichomonas vaginalis. The structure activity studies (SAR) revealed that the bis-uracil-isatin based conjugates were more effective than their corresponding mono conjugates in inhibiting the growth of T. vaginalis at approximately 10 μM with no visual effect on mammalian cells at the same concentration