Structure-Guided Discovery of New Antimicrobial Agents

A drug takes many years to develop and reach the market using the conventional drug discovery procedure. Computer-aided drug design (CADD) is an emerging technology that accelerates the process of drug discovery and minimizes the total expenditure associated with labour and resources. In the current scenario, the computational aided drug design (CADD) techniques play a significant role in the design and development of lead molecules for the treatment of various lethal pathological conditions. The prediction of the tertiary structure of a protein is a big concern in drug design and discovery. A typical drug discovery procedure starts with the tertiary structure of a protein. At present, a total of 184,407 protein structures are available in the protein data bank, which are determinedusing experimental methods. However, the procedures are difficult and time-consuming. A more advanced technique has been developed for the prediction of the 3D structure of a protein using a computational method. This technique has played a vital role in drug discovery. It has not only facilitated but also hastened the process of drug discovery. The method is named homology modeling since it involves the building of a protein model based on its homology to similar evolutionary proteins. The method is based on the fact that evolutionary related proteins have similar structures. In homology modelling, the 3D structure of a protein is derived from its primary sequence based on its similarity to the existing protein templates. There are many computational tools for homology modelling such as Modeller, Swiss model, Composer, 3D-JIGSAW, etc. The proposed book chapter will cover the introduction to homology modelling, step-by-step guide to building a protein model, various challenges and how to refine and validate the model, different algorithms related to sequence alignment, similarity search, and the applications of homology modelling in drug design and discovery. The chapter would be very fruitful to the readers to get insights into protein modelling, which will facilitate their research activities. It will be of great application in various disciplines,such as bioinformatics, physics, structural biology, and molecular biology. The content of the chapter will cover various research papers, review papers, and corresponding reference books.

Editorial overview: New protein production tools for structural biology

A model approach to accelerated drug discovery

Present at the flood: How structural biology came about, by Dickerson, Richard E.. 2005. Sinauer Associates, Sunderland, MA. 307 pp. $34.95 (paper) Robert M. Stroud*, * Department of Biochemistry, University of California at San Francisco, San Francisco, CA 94143, USA Present at the Flood: How Structural Biology Came About is an essential book for every structural biologist and a must for everyone who reflects on the origins of molecular biology. Present at the Flood, authored by Richard E. Dickerson, enchantingly traces the critical path to the discovery of the underlying atomic structures of proteins and DNA. These discoveries became the foundations of modern molecular biology. Present at the Flood interprets the 30 years that revolutionized structural and molecular biology, from 1933 to 1963, and is dedicated to Francis Crick. Francis also helped Dickerson to unearth some of the unpublished materials that illustrate the true history, such as the draft of the first erroneous DNA model. The moments of critical insight and of erroneous ideas are brilliantly captured by Dickerson, who was himself an essential, key ingredient in the discovery of the first protein structure (he was indeed “present at the flood”), and at the same institution where both the myoglobin and hemoglobin protein structures and the structure of DNA were discovered. These discoveries totally revolutionized biology. They were clearly synergistic with each other in a way that depended on the interplay between the personalities just as much as between structural thinkers. Present at the Flood reflects Dickerson's own total engagement and thrill in the process of discovery. It also reflects his keen recollection of the nuance, the slight remark that triggered a cascade of thought—the essence of some of the most important discoveries in all of biology. Like a detective novel, the book draws the reader into the fabric of discovery. It became a reality after the author presented a course of the same title in 2004 at UCLA. Dickerson retreads the pathway that he and colleagues in structural biology walked, carefully reasoning from the knowledge at the time to retrace the steps and ask precisely how insight came about. The book is very different from, for example, James Watson's personal perspective in The Double Helix; rather, it is a historical analysis of the ideas in the progression to discovery and how personalities augmented or bumped the process forward, backward, or sideways. Thus, he recounts mistakes in the early interpretations of Pauling and Corey and Crick and Watson, and recalls Erwin Chargaff's indignation after the DNA structure was determined. This all makes the historical analysis a personal encounter with the players themselves and a delight to read. Each of the first seven chapters begins with a hand-crafted Dickersonian pun! “In this life one should Perutz high goals, but ultimately settle for what one Kendrew”! Then, there follows a list of key publications, some of which are reprinted at the end. In total, over 40 of the original publications are reprinted, an invaluable record in itself. The text focuses onto Dickerson's interpretation of the meaning of research results, the flash of brilliance, the chance encounter, the stumbles, and communications that became the process of scientific discovery. Dickerson reflects colloquially on personalities, and their quirks that coupled to their contributions. The book reiterates the very words of the cast, including the sometimes sarcastic or even mean-spirited responses to competitors. Thus, the reader comes away knowing a lot about the people involved and the interplay of personality in discovery. Dickerson also questions the missed discoveries that could, or even should, have been made earlier—sometimes errors in logic, and happy accidents. The appendices present an invaluable guide to “Highly Recommended Readings,” and identifies those of key contributions, each with commentary as to why they are important milestones. Appendix 3 reflects on Irving Geis, the first artist and illustrator of the meaning of molecular structure who, with Dickerson, illustrated so many of the discoveries throughout the birth of structural biology and with whom Dickerson had a long very productive interaction. One of their books, The Proteins, coauthored by Dickerson and Geis (published in 1969), represented the first picture “from within” of what structural molecular biology revealed. It was also the beginning of the field of “computer graphic” displays of molecular structures. The Flood is dedicated to Francis H.C. Crick, whose contributions, like Dickerson's, were seminal both to the first discovery of the structure of proteins and to the discovery of the structure of DNA. While Crick worked with Perutz on the hemoglobin structure, Dickerson, in 1957, developed the mathematical basis and the key process to refine the “phases” of myoglobin, and thus, to determine its structure. As Crick realized the structure of DNA with Watson and Franklin's diffraction patterns in 1953, Dickerson, in 1980, was the first to determine the atomic structure of B-form DNA at atomic resolution. He recounts Crick's response on seeing Dickerson's atomic “B-form” structure: “So that's what it looks like!” Books written by investigators who shared in the discovery process often have a special intimacy to their story, and this book certainly is rich in this. Dickerson comments on Max Perutz, “Perutz turned out to be a gifted writer and essayist, which is more than many scientists can say.” Even more could be said of Dickerson's consummate ability to engage his readership! Few writers from the inside have been able to capture and analyze the process of discovery as well as Dickerson; he is scholarly, exact, balanced, and selfless. Dickerson's keen humor and insight draws the reader into the fun and excitement at the birth of molecular biology. Present at the Flood is important for the scientific record, as perhaps the only book that focuses on the discovery of protein structure seen in three-dimensional maps of linked atoms alongside and intertwined with the deduction of the structure of DNA. As such, it is a must for all who may reflect on the impact that structural biology has on molecular and cell biology, genetics, proteomics, biochemistry, or drug discovery.

High-resolution protein structures are essential for understanding biological mechanisms and drug discovery. While cryoEM has revolutionized structure determination of large protein complexes, most disease-related proteins are small (<50 kDa) and challenging to resolve due to low signal-to-noise ratios and alignment difficulties. Current scaffold protein strategies increase target size but suffer from inherent flexibility, resulting in poorly resolved targets compared to scaffolds. We present an iteratively engineered molecular design transforming antibody fragments (Fabs) into conformationally Rigid Fabs that enable high-resolution structure determination of small proteins (~20 kDa). This design introduces strategic disulfide bonds, creating well-folded, rigidly constrained Fabs applicable across various species, frameworks, and chimeric constructs. Rigid Fabs enabled high-resolution cryoEM structures (2.3-2.5 Å) of two small proteins: Ang2 (26 kDa) and KRAS (21 kDa). Our disulfide-constrained Rigid Fab strategy provides a general approach for overcoming target size limitation of single-particle cryoEM.

Background: Significant concerns are associated with the use of broad-spectrum antibacterial agents, including collateral eradication of beneficial bacteria from the human microbiome, the onset of antibacterial-associated infections, and continued emergence of antibacterial drug resistance. As such, a critical need for novel and selective antibacterial targets exists. The investigation of two such targets, each pertaining to the highly concerning infections caused by streptococcal species and Clostridioides difficile, are presented herein. Bacterial topoisomerase I represents a potentially promising narrowspectrum target as studies have arisen demonstrating its essentiality in bacterial species lacking the only other type IA topoisomerase (topoisomerase III). Additionally, recent studies demonstrating the essentiality of the fabK gene expressing enoyl-ACP reductase II (FabK) in C. difficile indicate its significant potential as a narrow-spectrum target. Presented here are data characterizing and validating both the TopoI and FabK enzymes as novel antibacterial targets via the implementation of an array of drug discovery techniques, including structural studies, biochemical assay development and application, and inhibitor screening and testing. Methods: An assortment of drug discovery techniques were employed for the targeting of SmTopoI and CdFabK, including different protein expression and purification techniques; X-ray crystallography; various biophysical and biochemical techniques for target characterization, validation, and drug screening; and different lead development and optimization studies. Results: The respective genes for SmTopoI and CdFabK have been cloned, and the expression and purification of various constructs of each target have been carried out and optimized for further analysis. The crystal structure of SmTopoI_N65 has been determined to 2.06 and diffracting CdFabK crystals (3.5 ) have been attained. A high-throughput plate-based biochemical fluorescence kinetic assay has been optimized for screening against the CdFabK enzyme. Furthermore, activity and modality of inhibition assessment of small-molecule inhibitors of the CdFabK enzyme have been conducted, including phenylimidazole and benzothiazole compounds. Phenylimidazole analogues have been found to display micromolar inhibitory activity against CdFabK, and a benzothiazole analogue has been found to display nanomolar inhibitory activity against the target. Conclusions: The SmTopoI and CdFabK enzymes present potentially novel, narrow-spectrum antibacterial drug targets, and substantial progress has been made toward the rational targeting of these two enzymes. Of particular note, the first structure of a Topo I fragment from a grampositive organism, S. mutans, has been determined. Enzymology and inhibitor studies have been conducted supporting the druggability of CdFabK and indicating the potential for selective inhibition of CdFabK.

Viruses with lipid envelopes must fuse their membranes with those of host cells to transfer their genomes and initiate infection. Depending on the virus, the membrane fusion process can occur at the plasma membrane, or at intracellular membranes following the internalization of virus particles. Not surprisingly, the viral proteins responsible for membrane fusion are highly diverse, as are the mechanisms by which the fusion processes can be triggered; however, common themes have emerged as our understanding of membrane fusion has developed. In particular, an expanding repertoire of high-resolution structures of viral fusion proteins (F proteins; VFPs) in pre- and postfusion conformations has driven the field forward. From these, we know that many VFPs undergo substantial conformational changes during fusion, forming highly stable rod-like structures to draw the membranes together (1⇓–3). Despite this, for VFPs other than influenza HA (4⇓⇓–7), high-resolution structures for all three major static conformations adopted during the virus life cycle, uncleaved prefusion, primed prefusion and postfusion, have remained incomplete. These limitations have hindered our development of insights into the fusion mechanism of viruses that, unlike influenza virus, enter cells at the plasma membrane at neutral pH. In a groundbreaking report in PNAS (8), Welch et al. solve the high-resolution X-ray crystal structure of the cleaved, prefusion form of the F protein of parainfluenza virus 5 (PIV5). This alters the state of affairs by providing an essential missing link in the understanding of paramyxovirus entry. In conjunction with previous work by these laboratories (9, 10), the new structure now affords us with a complete set of all three major static conformations of paramyxovirus F proteins (Fig. 1), a tour de force representing many years of effort …

In pharmaceutical research drug discovery and development process is timeconsuming and expensive. In many cases, it produces incompetent results due to the failure of in vitro and in vivo conventional approaches. Before any new drug is placed in the market it must undergo rigorous testing to get FDA approval. Due to the several limitations imposed by the drug discovery process, in recent times in silico approaches are widely applied in this field. The purpose of this review is to highlight the current molecular docking strategies used in drug discovery and to explore various advances in the field. In this review we have compiled database after an extensive literature search on docking studies which has found its applications relevant to the field of formulation and development. The papers retrieved were further screened to appraise the quality of work. In depth strategic analysis was carried out to confirm the credibility of the findings. The papers included in this review highlight the promising role of docking studies to overcome the challenges in formulation and development by emphasizing it's applications to predict drug excipient interactions which in turn assist to increase protein stability; to determine enzyme peptide interactions which maybe further used in drug development studies; to determine the most stable drug inclusion complex; to analyze structure at molecular level that ascertain an increase in solubility, dissolution and in turn the bioavailability of the drug; to design a dosage form that amplify the drug discovery and development process. This review summarizes recent findings of critical role played by molecular docking in the process of drug discovery and development. The application of docking approach will assist to design a dosage form in the most cost effective and time saving manner.

Inter-residue interactions play an essential role in driving protein folding, and analysis of these interactions increases our understanding of protein folding and stability and facilitates the development of tools for protein structure and function prediction. In this work, we systematically characterized the change of inter-residue interactions at various sequence separation cutoffs using two protein datasets. The first set included 100 diverse, nonredundant and high-resolution soluble protein structures, covering all four major structural classes, all-alpha, alpha/beta, alpha+beta, and all-beta; and the second set included 20 diverse, nonredundant and high-resolution membrane protein structures, representing 19 unique superfamilies. It was shown that the average number of inter-residue interactions in structures of both datasets displays the power-law behavior. Fitting parameters of the power-law function are directly related to the structural classes analyzed. These findings provided further insight into the distribution of short-, medium-, and long-range inter-residue interactions in both soluble and membrane proteins and could be used for protein structure prediction.

Membrane protein assemblies — towards atomic resolution analysis: Commentary

In drug discovery, determining the binding affinity and functional effects of small-molecule ligands on proteins is critical. Current computational methods can predict these protein–ligand interaction properties but often lose accuracy without high-resolution protein structures and falter in predicting functional effects. Here we introduce PSICHIC (PhySIcoCHemICal graph neural network), a framework incorporating physicochemical constraints to decode interaction fingerprints directly from sequence data alone. This enables PSICHIC to attain capabilities in decoding mechanisms underlying protein–ligand interactions, achieving state-of-the-art accuracy and interpretability. Trained on identical protein–ligand pairs without structural data, PSICHIC matched and even surpassed leading structure-based methods in binding-affinity prediction. In an experimental library screening for adenosine A1 receptor agonists, PSICHIC discerned functional effects effectively, ranking the sole novel agonist within the top three. PSICHIC’s interpretable fingerprints identified protein residues and ligand atoms involved in interactions, and helped in unveiling selectivity determinants of protein–ligand interaction. We foresee PSICHIC reshaping virtual screening and deepening our understanding of protein–ligand interactions. Predicting the binding affinity between small-molecule ligands and proteins is a key task in drug discovery; however, sequence-based methods are often less accurate than structure-based ones. Koh et al. develop a graph neural network using physicochemical constraints that discovers interactions between small molecules and proteins directly from sequence data and that can achieve state-of-the-art performance without the need for costly, experimental 3D structures.

PREFACE. 1 INTRODUCTION. 1.1 Aim of This Book. 1.2 An Overview of the Drug Discovery and Development Process. 1.3 The Pharmaceutical Industry. 1.4 Economics of Drug Discovery and Development. 1.5 Trends in Drug Discovery and Development. 1.6 Case Study #1. 1.7 Summary of Important Points. 1.8 Review Questions. 1.9 Brief Answers and Explanations. 1.10 Further Reading. 2 DRUG DISCOVERY: TARGETS AND RECEPTORS. 2.1 Drug Discovery Processes. 2.2 Medical Needs. 2.3 Target Identifi cation. 2.4 Target Validation. 2.5 Drug Interactions with Targets or Receptors. 2.6 Enzymes. 2.7 Receptors and Signal Transduction. 2.8 Assay Development. 2.9 Case Study #2. 2.10 Summary of Important Points. 2.11 Review Questions. 2.12 Brief Answers and Explanations. 2.13 Further Reading. 3 DRUG DISCOVERY: SMALL MOLECULE DRUGS. 3.1 Introduction. 3.2 Irrational Approach. 3.3 Rational Approach. 3.4 Antisense Approach. 3.5 RNA Interference Approach. 3.6 Chiral Drugs. 3.7 Closing Remarks. 3.8 Case Study #3. 3.9 Summary of Important Points. 3.10 Review Questions. 3.11 Brief Answers and Explanations. 3.12 Further Reading. 4 DRUG DISCOVERY: LARGE MOLECULE DRUGS. 4.1 Introduction. 4.2 Vaccines. 4.3 Antibodies. 4.4 Cytokines. 4.5 Hormones. 4.6 Gene Therapy. 4.7 Stem Cells and Cell Therapy. 4.8 Case Study #4. 4.9 Summary of Important Points. 4.10 Review Questions. 4.11 Brief Answers and Explanations. 4.12 Further Reading. 5 DRUG DEVELOPMENT AND PRECLINICAL STUDIES. 5.1 Introduction. 5.2 Pharmacodynamics. 5.3 Pharmacokinetics. 5.4 Toxicology. 5.5 Animal Tests, In Vitro Assays, and In Silico Methods. 5.6 Formulations and Delivery Systems. 5.7 Nanotechnology. 5.8 Case Study #5. 5.9 Summary of Important Points. 5.10 Review Questions. 5.11 Brief Answers and Explanations. 5.12 Further Reading. 6 CLINICAL TRIALS. 6.1 Defi nition of Clinical Trial. 6.2 Ethical Considerations. 6.3 Clinical Trials. 6.4 Regulatory Requirements for Clinical Trials. 6.5 Role of Regulatory Authorities. 6.6 Gene Therapy Clinical Trial. 6.7 Case Study #6. 6.8 Summary of Important Points. 6.9 Review Questions. 6.10 Brief Answers and Explanations. 6.11 Further Reading. 7 REGULATORY AUTHORITIES. 7.1 Role of Regulatory Authorities. 7.2 US Food and Drug Administration. 7.3 European Medicines Agency. 7.4 Japan's Ministry of Health, Labor and Welfare. 7.5 China's State Food and Drug Administration. 7.6 India's Central Drugs Standard Control Organization. 7.7 Australia's Therapeutics Goods Administration. 7.8 Canada's Health Canada. 7.9 Other Regulatory Authorities. 7.10 Authorities Other than Drug Regulatory Agencies. 7.11 International Conference on Harmonization. 7.12 World Health Organization. 7.13 Pharmaceutical Inspection Cooperation Scheme. 7.14 Case Study #7. 7.15 Summary of Important Points. 7.16 Review Questions. 7.17 Brief Answers and Explanations. 7.18 Further Reading. 8 REGULATORY APPLICATIONS. 8.1 Introduction. 8.2 Food and Drug Administration. 8.3 European Union. 8.4 Japan. 8.5 China. 8.6 India. 8.7 Australia. 8.8 Canada. 8.9 Case Study #8. 8.10 Summary of Important Points. 8.11 Review Questions. 8.12 Brief Answers and Explanations. 8.13 Further Reading. 9 GOOD MANUFACTURING PRACTICE: REGULATORY REQUIREMENT. 9.1 Introduction. 9.2 United States. 9.3 Europe. 9.4 International Conference on Harmonization. 9.5 Core Elements of GMP. 9.6 Selected GMP Systems. 9.7 The FDA's New cGMP Initiative. 9.8 Case Study #9. 9.9 Summary of Important Points. 9.10 Review Questions. 9.11 Brief Answers and Explanations. 9.12 Further Reading. 10 GOOD MANUFACTURING PRACTICE: DRUG MANUFACTURING. 10.1 Introduction. 10.2 GMP Manufacturing. 10.3 GMP Inspection. 10.4 Manufacture of Small Molecule APIs (Chemical Synthesis Methods). 10.5 Manufacture of Large Molecule APIs (Recombirant DNA Methods). 10.6 Finished Dosage Forms. 11 FUTURE PERSPECTIVES. 11.1 Past Advances and Future Challenges. 11.2 Small Molecule Pharmaceutical Drugs. 11.3 Large Molecule Biopharmaceutical Drugs. 11.4 Traditional Medicine. 11.5 Individualized Medicine. APPENDIX 1 HISTORY OF DRUG DISCOVERY AND DEVELOPMENT. A1.1 Early History of Medicine. A1.2 Drug Discovery and Development in the Middle Ages. A1.3 Foundation of Current Drug Discovery and Development. A1.4 Beginnings of Modern Pharmaceutical Industry. A1.5 Evolution of Drug Products. A1.6 Further Reading. APPENDIX 2 CELLS, NUCLEIC ACIDS, GENES, AND PROTEINS. A2.1 Cells. A2.2 Nucleic Acids. A2.3 Genes and Proteins. A2.4 Further Reading. APPENDIX 3 SELECTED DRUGS AND THEIR MECHANISMS OF ACTION. APPENDIX 4 A DHFR PLASMID VECTOR. APPENDIX 5 VACCINE PRODUCTION METHODS. APPENDIX 6 PHARMACOLOGY/ TOXICOLOGY. REVIEW FORMAT. APPENDIX 7 EXAMPLES OF GENERAL BIOMARKERS. APPENDIX 8 TOXICITY GRADING. APPENDIX 9 HEALTH SYSTEMS IN SELECTED COUNTRIES. ACRONYMS. GLOSSARY. INDEX.

Chapter 5 - Homology modeling: Developing 3D structures of target proteins missing in databases

Molecular modeling of proteins including homology modeling, structure determination, and knowledge-based protein design requires tools to evaluate and refine three-dimensional protein structures. Steric clash is one of the artifacts prevalent in low-resolution structures and homology models. Steric clashes arise due to the unnatural overlap of any two nonbonding atoms in a protein structure. Usually, removal of severe steric clashes in some structures is challenging since many existing refinement programs do not accept structures with severe steric clashes. Here, we present a quantitative approach of identifying steric clashes in proteins by defining clashes based on the Van der Waals repulsion energy of the clashing atoms. We also define a metric for quantitative estimation of the severity of clashes in proteins by performing statistical analysis of clashes in high-resolution protein structures. We describe a rapid, automated, and robust protocol, Chiron, which efficiently resolves severe clashes in low-resolution structures and homology models with minimal perturbation in the protein backbone. Benchmark studies highlight the efficiency and robustness of Chiron compared with other widely used methods. We provide Chiron as an automated web server to evaluate and resolve clashes in protein structures that can be further used for more accurate protein design.

Antimicrobial resistance is at an all-time high and new drugs are required to overcome this crisis. Traditional approaches to drug discovery have failed to produce novel classes of antibiotics, with only a few currently in development. It is thought that novel classes will come from antibacterial drug discovery efforts that focus on unconventional targets. One such collection of antibacterial targets are those that comprise central carbon metabolism. Targets of this kind have been largely overlooked because conventional antibacterial testing media are ill-suited for exploring carbon source utilization. Nevertheless, as a consequence of infection, bacteria must find a carbon source in order to survive. Here, we review what is known about the carbon sources available and used by bacteria in different host infection sites. We also look at discovery efforts targeting central carbon metabolism and evaluate how these processes can influence antibiotic efficacy.