Elif Ozkirimli Olmez

Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.

Research Article: Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening

Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design.

Protein-Peptide Interactions Revolutionize Drug Development

Protein-protein interactions form the basis of many cellular processes. Disruption or deregulation of these complex interactions is the main cause of a significant number of human ailments. Consequently, there is intense research effort to design inhibitors that target specific protein-protein interactions. This places intricate protein-protein interactions in the heart of the development for novel drug leads. The emergence of ‘omic’ technologies, namely genomics, transcriptomics and proteomics, has greatly accelerated our understanding of the protein-protein interaction networks leading to the discovery of a number of proteins and their interaction interface as potential drug targets.

Investigation of TEM-1 and SHV-1 beta-lactamase ligand binding

The abuse, overuse and misuse of beta-lactam antibiotics in treating bacterial infections have caused bacteria to develop resistance against them. One common antibiotic resistance mechanism utilized by bacteria is the production of beta-lactamase enzymes that cleave the amide bond in beta-lactam ring rendering the antibiotic ineffective. One way to combat this problem is to use beta-lactamase inhibitors in combination with beta-lactam antibiotics. Beta-lactamase inhibitor protein (BLIP) is an effective inhibitor of class A beta-lactamases such as TEM-1 and SHV-1. In the current research, the binding of BLIP to TEM-1 and to SHV-1 beta lactamase was investigated using molecular dynamics simulations. The binding free energies of BLIP complex with TEM-1 and SHV-1 betalactamases were calculated using Molecular Mechanic Poisson Bolztmann Surface Area (MM-PBSA) methodology. It was found that BLIP has significant differences in binding affinities toward TEM-1 and SHV-1 beta-lactamases.

An evolutionarily conserved allosteric site modulates beta-lactamase activity

Abstract Declining efficiency of antibiotic-inhibitor combinatorial therapies in treating beta-lactamase mediated resistance necessitates novel inhibitor development. Allosteric inhibition offers an alternative to conventional drugs that target the conserved active site. Here, we show that the evolutionarily conserved PWP triad located at the N-terminus of the H10 helix directly interacts with the allosteric site in TEM-1 beta-lactamase and regulates its activity. While point mutations in the PWP triad preserve the overall secondary structures around the allosteric site, they result in a more open and dynamic global structure with decreased chemical stability and increased aggregation propensity. These mutant enzymes with a less compact hydrophobic core around the allosteric site displayed significant activity loss. Detailed sequence and structure conservation analyses revealed that the PWP triad is an evolutionarily conserved motif unique to class A beta-lactamases aligning its allosteric site and hence is an effective potential target for enzyme regulation and selective drug design.

Aminopurine derivatives as putative SopE inhibitors

Abstract Salmonella outer protein E, SopE, is a virulence factor that is secreted from gram-negative Salmonella enterica, which is pathogenic to humans and is responsible for gastroenteritis and typhoid fever. SopE targets the Rho GTPase family proteins of the host cell and manipulates them by mimicking GTPase regulatory protein guanine nucleotide exchange factor. The aim of this work is the investigation of novel inhibitors against SopE. Structure-based pharmacophore modeling was used to identify structural features that would be important for SopE recognition. Glide fragment library was used for four-point pharmacophore hypothesis development. Small-molecule database screening was performed based on a 3D similarity to the best pharmacophore hypothesis. Binding affinity of filtered database molecules to SopE was predicted quantitatively by molecular docking and scoring using Glide software. Top scoring hits were further analyzed and five molecules were proposed as potent and selective SopE inhibitors. Four out of five proposed molecules were found to be aminopurine derivatives.

Discovery of YopE Inhibitors by Pharmacophore-Based Virtual Screening and Docking.

Gram-negative bacteria Yersinia secrete virulence factors that invade eukaryotic cells via type III secretion system. One particular virulence member, Yersinia outer protein E (YopE), targets Rho family of small GTPases by mimicking regulator GAP protein activity, and its secretion mainly induces cytoskeletal disruption and depolymerization of actin stress fibers within the host cell. In this work, potent drug-like inhibitors of YopE are investigated with virtual screening approaches. More than 500,000 unique small molecules from ZINC database were screened with a five-point pharmacophore, comprising three hydrogen acceptors, one hydrogen donor, and one ring, and derived from different salicylidene acylhydrazides. Binding modes and features of these molecules were investigated with a multistep molecular docking approach using Glide software. Virtual screening hits were further analyzed based on their docking score, chemical similarity, pharmacokinetic properties, and the key Arg144 interaction along with other active site residue interactions with the receptor. As a final outcome, a diverse set of ligands with inhibitory potential were proposed.

Dynamic analysis of ß lactamase ligand interaction

β-lactam antibiotics are the most commonly used antibiotics which cause bacterial cell lysis by inhibiting the enzyme responsible for the cell wall synthesis. Production of β-lactamase enzyme, which catalyzes the hydrolysis of β-lactam ring of β-lactam antibiotics is the most common mechanism of bacterial resistance. β-Lactamase Inhibitory Protein (BLIP), is an effective inhibitor of class A β-lactamases such as TEM-1 and SHV-1. TEM-1 and SHV-1 are the most commonly found β-lactamases and they are responsible for the resistance to β-lactam antibiotics of various pathogenic bacteria. In an effort to elucidate the mechanism of β-lactamase inhibiton by BLIP and to make predictions of binding affinity between these molecules, Molecular Dynamics (MD) simulations were performed on TEM-1 and SHV-1 bound to BLIP and BLIP based peptides. Asp49 residue which is known to play a critical role on binding on BLIP was mutated to Alanine to determine the contribution of this residue to binding. Binding free energy of the TEM-1 and SHV-1 bound BLIP, mutant BLIP (D49A) complexes were estimated by the molecular mechanics Poisson Boltzmann Surface Area method (MM-PBSA). Free energy of binding calculations show that the mutation on D49 causes a decrease in binding affinity for both TEM-1 and SHV-1 β-lactamase.

Computational prediction of protein-protein interactions in sphingolipid signaling network

Proteins carry out most of the work in the cell such as immunological recognition, DNA repair and replication, enzymatic activity, cell signaling by interacting with other proteins. Therefore, elucidation of the protein-protein interaction network will assist in understanding molecular mechanism of cellular activities. Recent advances in high-throughput experimental methods have provided a large amount of data that need to be sorted and interpreted to find biologically relevant interactions and pathways. In silico methods that can accurately predict properties of protein-protein interactions have gained increased interest. In this study, the network of sphingolipid (SL) signaling proteins was constructed using computational prediction methods to contribute to missing interactions among the components of sphingolipid protein-protein interaction (PPI) network. As a result of the studies by our group, the potential protein interactions between YER019W-YHL020C and YGR143W-YKL126W were identified. The new predictions proposed by this research can guide rational design of new experiments.