Revealing the Mechanism of Thiopeptide Antibiotics at Atomistic Resolution : Implications for Rational Drug Design

For decades drug design has primarily focused on small molecules that bind to well-formed tight binding pockets, such as the catalytic centers of enzymes. Recently, there is increasing interest to design compounds that disrupt or stabilize biomacromolecular interfaces (e.g. protein–protein, protein–DNA, protein–RNA, protein–lipid interfaces). These non-traditional drug targets hold great therapeutic potential as they govern cellular pathways. In contrast to traditional drug targets, where computational methods are now routinely and productively used to complement experiments, the use of computer-based approaches for the study and design of interfacial modulators is still in its infancy. The current thesis is a first detailed study into understanding the effects of modulators of a protein–RNA interface and developing computer-based approaches for their design. This work focuses on the 23S-L11 subunit of the ribosomal GTPase-associated region (GAR), a prototypic protein–RNA interface of high relevance in the development of novel antibacterials. The GAR is the target of naturally occuring thiopeptide antibiotics. These unique molecules are effective inhibitors of bacterial protein synthesis, but are currently unused in human antibacterial therapy due to their low aqueous solubility. Their mechanism of action is explored in the current thesis, enabling the design and proposition of new chemical scaffolds targeting their binding site. The specific challenges associated with the 23-SL11-thiopeptide system, such as the inherent flexibility of the protein–RNA composite environment and the size and structural complexity of the thiopeptide ligands, are addressed by a combination of computational chemistry approaches at different levels of granularity and a steady feedback with experimental data to validate and improve the computational techniques. These approaches range from quantummechanics for deriving optimized intramolecular parameters and partial atomic charges for the thiopeptide compounds, to molecular dynamics simulations accounting for the binding site’s flexibility, to molecular docking studies for predicting the binding modes of different thiopeptides and derivatives. All-atom molecular dynamics simulations were conducted, providing a detailed understanding of the effect of thiopeptide binding at a previously unmet resolution. The findings of this work, coupled with previous experimental knowledge, strongly support the hypothesis that restricting the binding site’s conformational flexibility is an important component of the thiopeptide antibiotics’ mode of action. With the help of an MD-docking-MD workflow and an energy decomposition analysis crucial residues of the binding site and pharmacologically relevant moieties within the ligand structures could be identified. A 4D-pharmacophore model is presented that was derived from a refined 23S-L11-thiopeptide complex and additionally accounts for the dynamic stability of molecular interactions formed between the antibiotic and the ribosomal binding site as the fourth dimension. The results of this thesis revealed, for the first time, a plausable description of the thiopeptide antibiotics’ mode of action, down to the details of their pharmacologically relevant parts and provide a computational framework for the design of new ligands

Flexible Receptor Docking Method Development and Molecular Dynamics Studies Towards Targeting Dynamic Protein Surfaces.

Protein-protein interactions are integral for cellular function, playing a huge role in processes such as cell signaling and transcription regulation. Targeting these essential interactions with small molecule inhibitors is important from a biochemical and pharmaceutical perspective. This dissertation contains chapters on multidisciplinary, collaborative approaches to investigate transcription regulation as well as MHC Class I assembly, which is involved in the immune response. During these projects I applied a variety of computational tools and developed a new docking methodology in CHARMM (CDOCKER). This new version of CDOCKER incorporates receptor flexibility through maintaining selected side-chains in an all-atom representation, while the rest of the receptor is represented as a grid. This version of CDOCKER includes a newly implemented sampling protocol that leads to docking accuracy that is competitive with and even exceeds that of other commonly used docking software in redocking trials. This docking methodology was applied to identify a putative ATP binding on calreticulin (CRT), a chaperone key to MHC Class I assembly and the immune response. This work was a collaborative effort with the Raghavan research group at the University of Michigan and was the first demonstration that CRT both binds and catalyzes ATP. We added further automated functionality to the CDOCKER method to investigate small- molecules covalently bound to receptors in collaboration with the Mapp research group at the University of Michigan. The tethering method was able to stabilize the dynamic surface of GACKIX for crystallization and modeled small-molecules that were identified experimentally but were unable to be crystalized. Finally, we employed Gō-like models to investigate the allosteric signaling between the two binding sites on GACKIX. These studies demonstrated the positive allostery arises from the first peptide paying the entropic cost of binding for the second peptide. The developments in docking methodology within CHARMM allow for targeting of fluid receptors such as GACKIX. A multidisciplinary approach to investigate complex cellular processes such as transcription regulation or the immune response takes advantage of the strengths of the different approaches and leads to advancements in understanding of the process at different size scales, atomistic to in vitro and even in vivo.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116710/1/gagnonj_1.pd

Dynamics of protein-drug interactions inferred from structural ensembles and physics-based models

The conformational flexibility of target proteins is a major challenge in understanding and modeling protein-drug interactions. A fundamental issue, yet to be clarified, is whether the observed conformational changes are controlled by the protein, or induced by the inhibitor. While the concept of induced fit has been widely adopted for describing the structural changes that accompany ligand binding, there is growing evidence in support of the dominance of proteins' intrinsic dynamics, which has been evolutionarily optimized to accommodate its functional interactions. The wealth of structural data for target proteins in the presence of different ligands now permits us to make a critical assessment of the balance between these two effects in selecting the bound forms. We focused on three widely studied drug targets, HIV-1 reverse transcriptase, p38 MAP kinase, and cyclin-dependent kinase 2. A total of 292 structures determined for these enzymes in the presence of different inhibitors as well as unbound form permitted us to perform an extensive comparative analysis of the conformational space accessed upon ligand binding, and its relation to the intrinsic dynamics prior to ligand binding as predicted by elastic network model analysis. Further, we analyzed NMR ensembles of ubiquitin and calmodulin representing their microseconds range solution dynamics. Our results show that the ligand selects the conformer that best matches its structural and dynamic properties amongst the conformers intrinsically accessible to the protein in the unliganded form. The results suggest that simple but robust rules encoded in the protein structure play a dominant role in pre-defining the mechanisms of ligand binding, which may be advantageously exploited in designing inhibitors. We apply these lessons to the study of MAP kinase phosphatases (MKPs), which are therapeutically relevant but challenging signaling enzymes. Our study provides insights into the interactions and selectivity of MKP inhibitors and shows how an allosteric inhibition mechanism holds for a recently discovered inhibitor of MKP-3. We also provide evidence for the functional significance of the structure-encoded dynamics of rhodopsin and nicotinic acetylcholine receptor, members of two membrane proteins classes serving as targets for more than 40% of all current FDA approved drugs

Evidence for the Involvement of the Chemosensory Protein AgosCSP5 in Resistance to Insecticides in the Cotton Aphid, Aphis gossypii

It has been speculated that insect chemosensory proteins (CSPs) may have additional roles beyond olfaction. In this study, the phylogenetic and genomic analyses of the CSPs of the cotton aphid, Aphis gossypii, revealed the presence of gene gain-and-loss among different aphid field populations. Differential expressions of eight CSP genes were demonstrated after treatments with insecticides of different modes of action. The expression of AgosCSP5 was significantly upregulated by the insecticide treatments in a dose-dependent manner. The Drosophila flies overexpressing AgosCSP5 were significantly less susceptible to the insecticides, omethoate, imidacloprid and cypermethrin but not to deltamethrin and tau-fluvalinate, compared with control flies. The transgenic Drosophila flies exhibited an LC50 resistance ratio of 2.6 to omethoate, compared with control flies. Likewise, the mortality of the transgenic flies to imidacloprid and cypermethrin was significantly lower than that of the control flies (p < 0.01). Homology modelling, molecular docking and dynamic simulation supported the interactions and revealed a higher stability of AgosCSP5/insecticide complexes than AgosCSP5/semiochemical complexes. Our study demonstrates for first time the in vivo evidence for the involvement of CSP genes in insecticide resistance of crop insect pests and provides new insights of the newly discovered CSP-mediated insect resistance mechanism to insecticides

Accounting for Large Amplitude Protein Deformation during in Silico Macromolecular Docking

Rapid progress of theoretical methods and computer calculation resources has turned in silico methods into a conceivable tool to predict the 3D structure of macromolecular assemblages, starting from the structure of their separate elements. Still, some classes of complexes represent a real challenge for macromolecular docking methods. In these complexes, protein parts like loops or domains undergo large amplitude deformations upon association, thus remodeling the surface accessible to the partner protein or DNA. We discuss the problems linked with managing such rearrangements in docking methods and we review strategies that are presently being explored, as well as their limitations and success

Merging Ligand-Based and Structure-Based Methods in Drug Discovery: An Overview of Combined Virtual Screening Approaches

Virtual screening (VS) is an outstanding cornerstone in the drug discovery pipeline. A variety of computational approaches, which are generally classified as ligand-based (LB) and structure-based (SB) techniques, exploit key structural and physicochemical properties of ligands and targets to enable the screening of virtual libraries in the search of active compounds. Though LB and SB methods have found widespread application in the discovery of novel drug-like candidates, their complementary natures have stimulated continued e orts toward the development of hybrid strategies that combine LB and SB techniques, integrating them in a holistic computational framework that exploits the available information of both ligand and target to enhance the success of drug discovery projects. In this review, we analyze the main strategies and concepts that have emerged in the last years for defining hybrid LB + SB computational schemes in VS studies. Particularly, attention is focused on the combination of molecular similarity and docking, illustrating them with selected applications taken from the literature

Psychology Case Record

UNDIFFERENTIATED SCHIZOPHRENIA: Mrs. M. was apparently normal 1½ years back, she was staying in her home, and carrying out her routine work. Her family members noticed that gradually she becomes dull and withdrawn and preoccupied, she started avoiding to interact with family members and in work place. And also she was found to be talking and laughing to herself. She would sit alone near the tree and would talk to herself. For which if she was questioned she would not reply anything. Her sleep is disturbed they noticed the frequent awakenings is night, she most of the time, she was found to be talking to herself in the night, she would spend most of the time alone not interacting with others. She stopped going for work. Not showing interest in doing house hold work and not maintaining herself. She would not show interest in taking care of her husband who was ill for past 2-3 yrs. 2 months later the family members have brought her to magico religious treatment. But the symptoms persisted. Her husband became very sick and hospitalised. But she did not visit him atleast once, though her husband wished to meet her in the terminal stage of illness. Her children forced her to meet him, but she refused, she didn't give any reason for that. Her husband Mr. Elumalai died in August 2009. Due to alcoholic liver disease. The day of her husband's death she did not worry about it, she did not show the normal emotional response for the death. She refused to participate in the formalities in the death ceremony. She did not interact with any relatives who visited the ceremony. She was found preoccupied and talking to herself. 16th day of death ceremony she thrown her husband's photo into the well and shouted that person is not her husband. PARANOID SCHIZOPHRENIA: Patient was apparently normal about 1½ years back living with his parents working as salesman in a private company and having good interpersonal relationship. Once he had a fight with people in a neighbourhood house regarding disposal of sewage which resulted in physical assault. The neighbour had threatened that he had political influence and would harm him. From then on the patient started expressing fear that the neighbor would harm him. Next day he and apologized to the neighbour and the issue was resolved by talks and the neighbour also apologized in return. But the patient was not convinced and he started saying that the neighbor was still trying to harm him. He used to tell that the neighbor is making gestures to several people indicating that he should be followed. The patient used to tell that wherever he went he was being followed by people set up by the neighbor to follow him and to report his activities. He started saying that he could hear voices of several unknown males and that of his neighbor discussing among themselves the different ways to harm him. He didn’t leave his room claiming that he would harmed and discontinued going to work. Gradually he started to bath and change his clothes less frequently about once in 2 to 3 days. He used to remain preoccupied and at times start shouting that people are coming to harm him and beg his parents to save him from them. He slept only about 4 to 5 hours in a day pacing inside the room or sitting in a place preoccupied. After one year of such behavioural disturbance he was taken to private psychiatrist and treated with tab.Risperidone 2 mg 1-0-2, tab.Benzhexol 2 mg 1-0-1 and tab.Diazepam 5 mg 0-0-2.With treatment his suspiciousness, hearing voices,sleep and self care improved within 6 to 7 months. He discontinued the drugs claiming that he was feeling tired and drowsy always. He didn’t go for work. For the past 6 months it was noticed that he was frequently washing his hands and on enquiry the patient explained that he felt when he touched certain articles in bathroom whether he would get the germs present in the objects. Gradually he started to wash his hands for about 10 to 15 minutes every time he touched any object in the house. He used to spend about 7 to 8 hours in a day involved in washing. For past 1 month he started saying that the neighbor was incorporating the germs in the objects so that he would be affected by life threatening illness. He started saying that the neighbor was coming to know of all his thoughts through by some unknown mechanism and gesturing at him in a mocking way. His sleep and self care also reduced. He was brought to IMH for treatment. No history of persistent sad mood / crying spells / suicidal ideas. No history of elated mood / excessive spending / tall claims. No history of substance abuse. BIPOLAR AFFECTIVE DISORDERMANIA WITH PSYCHOTIC FEATURES: Patient was apparently normal about 6 months back living with her daughter and having good interpersonal relationship. When it was noticed by the informant that the patient was talking excessively than before even with unknown people about various issues like politics, religions and that she had visited several foreign places. When enquired why she was telling lies she would laugh and say that she was simply playing with others. Gradually she started becoming easily irritated and would demand that she wanted several varieties of food to eat and that everyone should watch the TV programs that she wanted to watch. She would demand money about 1,000 to 2,000 rupees daily saying that she wanted to eat in hotels. She slept only for 2 to 3 hours in night and would spend the remaining time in watching TV in loud volume and sing songs. She started saying that she was the creator of the world and that she can destroy it in a second if she gets angry. She started saying that because of her powers some bad people were trying to kill her and continue with their bad works. As she started to assault others claiming that they were coming to harm her she was brought for treatment about 1 month back and started on T.sodium valproate 200 mg 2-2-2,T.Risperidone 2 mg 1-0-1 and T.Diazepam 5 mg 0-0-2. With treatment her symptoms has reduced in intensity. No history of sad mood / crying spells / suicidal ideas / suicidal gestures. No history of hearing voices / seeing images. No history of thoughts being known to others / being controlled by others. No history of substance use. No history of fever / head injury / seizures. MENTAL SUBNORMALITY ADJUSTMENT DISORDER: Mr. V 18 years old by apparently normal 3 years back. He started consuming alcohol introduced by his friends while he failed 10th standard. He started with 150 ml beer and developed tolerance upto 1800 – 2400 ml. He stopped alcohol for 2 months due to headache and vomiting. To overcome these affects he started smoking cigarate excessively. He had an affair with a girl, he was fully preoccupaid about her, he used to think about her atleast 20 hours in a day. So he could not able to concentrate in his study. He failed in +2. He was troubled by the girls brother, who apposed their love. Each of them fought. Issue was taken to police station. Past one year his parents noticed he has disturbed sleep, he falls in sleep around 2.30 a.m. slept for 4 hours. He was unable to concentrate in his studies. He keeps on think about her parents arguments. He could not go to sleep whole the night. His parents reported that he becomes disobedient not returning home in time. And spends all the time with his friends. He roaming purposeless. If they ask about him. He become irritable and abusing. He has runout from house twice in the past two years. Whenever he was scolded by his parents. He would become irritable and restless and make injurius in his body, like burning his arm, making cutting with sharp instruments. No H/O head injury/ LOC/ ENT Bleeding. No history of lauging to self / talking to self. No H/O crying spells. No H/O inflated self esteem. No H/O social problem. No H/O debts. Legal complication + No history of suicidal, ideation are attempt. No H/O of thought insertion / withdrawal and broadcasting. Nil Medical and Psychatric History. CHRONIC SCHIZOPHRENIA: Mr. G was apparently normal 20 years back. He has completed +2 after 4 attempts. He joint typewriting (lower) at the time he was dull and withdrawn. He was laughing to self at times. When question he was evasive. He showed lake of interest in his work. He was very lethargic. Considering his poor scholastic performance, his parents did not force him to pursue his studies further. He remained at home and spent his time watching TV. He begans to express suspicious ideas that somebody is watching him. Observing his actions. He began to talk to self. His father them took him is a neurologist and was giving him T. Hexidol tds for 2 years on the advise of neurologist. Then he stopped taking medications for the past 15 years. 2 years back he became very dull. He became poorly communicative. He did not interact with other family members. He had to be forced to take bath and to maintain her personal hygient when he is not forced, he would stay the same and would remain unclean 6 months before her father on the advice of one of his friends to him to SCARF foundation. He was receiving the drugs, he involves himself in some work has adequate sleep. He has been brought by his father to IMH to transfer his mother pension to him so that it would help him in future. No H/O suicidal attempts / crying to self. No H/O seizures. No H/O head injury. No H/O infection