Chemoinformatic Identification of Novel Inhibitors against Mycobacterium tuberculosis L-aspartate α-decarboxylase

L-Aspartate α-decarboxylase (ADC) belongs to a class of pyruvoyl dependent enzymes and catalyzes the conversion of aspartate to β-alanine in the pantothenate pathway, which is critical for the growth of several micro-organisms, including Mycobacterium tuberculosis (Mtb). Its presence only in micro-organisms, fungi and plants and its absence in animals, particularly human, make it a promising drug target. We have followed a chemoinformatics-based approach to identify potential drug-like inhibitors against Mycobacterium tuberculosis L-aspartate α-decarboxylase (MtbADC). The structure-based high throughput virtual screening (HTVS) mode of the Glide program was used to screen 333,761 molecules of the Maybridge, National Cancer Institute (NCI) and Food and Drug Administration (FDA) approved drugs databases. Ligands were rejected if they cross-reacted with S-adenosylmethionine (SAM) decarboxylase, a human pyruvoyl dependent enzyme. The lead molecules were further analyzed for physicochemical and pharmacokinetic parameters, based on Lipinski's rule of five, and ADMET (absorption, distribution, metabolism, excretion and toxicity) properties. This analysis resulted in eight small potential drug-like inhibitors that are in agreement with the binding poses of the crystallographic ADC:fumarate and ADC:isoasparagine complex structures and whose backbone scaffolds seem to be suitable for further experimental studies in therapeutic development against tuberculosis

Validation of Drug-Like Inhibitors against Mycobacterium Tuberculosis L-Aspartate α-Decarboxylase Using Nuclear Magnetic Resonance (1H NMR)

10.1371/journal.pone.0045947PLoS ONE79

Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors

Investigating the molecular basis of Siah1 and Siah2 E3 ubiquitin ligase substrate specificity.

The Siah1 and Siah2 E3 ubiquitin ligases play an important role in diverse signaling pathways and have been shown to be deregulated in cancer. The human Siah1 and Siah2 isoforms share high sequence similarity but possess contrary roles in cancer, with Siah1 more often acting as a tumor suppressor while Siah2 functions as a proto-oncogene. The different function of Siah1 and Siah2 in cancer is likely due to the ubiquitination of distinct substrates. Hence, we decided to investigate the molecular basis of the substrate specificity, utilizing the well-characterized Siah2 substrate PHD3. Using chimeric and mutational approaches, we identified critical residues in Siah2 that promote substrate specificity. Thus, we have found that four residues in the N-terminal region of the Siah2 substrate binding domain (SBD) (Ser132, His150, Pro155, Tyr163) are critical for substrate specificity. In the C-terminal region of the SBD, a single residue, Leu250, was identified to promote the specific binding of Siah2 SBD to PHD3. Our study may help to overcome the challenges in the identification of Siah2 specific inhibitors

Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3.

<p>(<b>a</b>) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1]^NT[S2]^CT and SBD[S1]^NT[S2]^CT(bottom panel). The original residue numbers are labeled in the respective colors (<b>b</b>) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)]^NT[S2]^CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2]^NT[S1-(Q121L/T160A]^CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.</p

Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3.

<p>(<b>a</b>) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. (<b>b</b>) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut]^NT[S2]^CT mutant increased binding compared to [S1-6Mut]^NT[S2]^CT. (<b>c</b>) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p<0.05.</p

Interaction of Siah2 with PHD3.

<p>(<b>a</b>) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106547#s2" target="_blank">Materials and Methods</a>”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. (<b>b</b>) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106547#s2" target="_blank">Material and Methods</a>”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.</p

Siah1 exhibits weak binding compared to Siah2 with PHD3.

<p>HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. (<b>a</b>) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 (<b>b</b>) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.</p

Docking model of the N-terminal Siah2 SBD and PHD3.

<p>N-terminal region (1–100) of the modeled Siah2 SBD was docked with PHD3 using an automated Cluspro server. The complex was then presented using Pymol as (<b>a</b>) cartoon representation, and (<b>b</b>) space filling representation. (<b>c</b>) The details of the interactions were obtained by PDBsum. The number of H-bond lines between any two residues indicates the number of potential hydrogen bonds between them. For non-bonded contacts, the width of the striped line is proportional to the number of atomic contacts.</p