Roles of Virtual Screening and Molecular Dynamics Simulations in Discovering and Understanding Antimalarial Drugs.

Malaria continues to be a global health threat, with approximately 247 million cases worldwide. Despite therapeutic interventions being available, patient compliance is a problem due to the length of treatment. Moreover, drug-resistant strains have emerged over the years, necessitating urgent identification of novel and more potent treatments. Given that traditional drug discovery often requires a great deal of time and resources, most drug discovery efforts now use computational methods. In silico techniques such as quantitative structure-activity relationship (QSAR), docking, and molecular dynamics (MD) can be used to study protein-ligand interactions and determine the potency and safety profile of a set of candidate compounds to help prioritize those tested using assays and animal models. This paper provides an overview of antimalarial drug discovery and the application of computational methods in identifying candidate inhibitors and elucidating their potential mechanisms of action. We conclude with the continued challenges and future perspectives in the field of antimalarial drug discovery.

Antimicrobial Peptides and Copper(II) Ions: Novel Therapeutic Opportunities.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Molecular Dynamics Investigation into the Effect of Zinc(II) on the Structure and Membrane Interactions of the Antimicrobial Peptide Clavanin A.

Clavanin A (ClavA) is an antimicrobial peptide (AMP) whose antimicrobial activity is enhanced in the presence of Zn(II) ions. The antimicrobial activity of ClavA has been shown to increase 16-fold in the presence of Zn(II) ions. In this study, we investigate the potential sources of this enhancement, namely, the effect of Zn(II) binding on the helical conformation of ClavA and on the ClavA interaction with a model for gram-negative bacterial membranes. In addition, we investigate the effect of Zn(II) on the membrane mechanical properties. We employed all-atom equilibrium molecular dynamics simulations initiated from both fully helical and random coil structures of ClavA. We observe that Zn(II) can stabilize an existing helical conformation in the Zn(II)-binding region, but we do not observe induction of helical conformations in systems initiated in random coil configurations. Zn(II) binding to ClavA provides more favorable electrostatics for membrane association in the C-terminal region. This is evidenced by longer and stronger C-terminal-lipid interactions. Zn(II) is also capable of modulating the membrane properties in a manner which favors ClavA insertion and the potential for enhanced translocation into the cell. This work provides insights into the role of divalent metal cations in the antimicrobial activity of ClavA. This information can be used for the development of synthetic AMPs containing motifs that can bind metals (metalloAMPs) for therapeutic and medical purposes.

Single Chain Polymeric Nanoparticles to Promote Selective Hydroxylation Reactions of Phenol Catalyzed by Copper.

Metal-containing single chain polymeric nanoparticles (SCPNs) can be used as synthetic mimics of metalloenzymes. Currently, the role of the folded polymer backbones on the activity and selectivity of metal sites is not clear. Herein, we report our findings on how polymeric frameworks modulate the coordination of Cu sites and the catalytic activity/selectivity of Cu-containing SCPNs mimicking monophenol hydroxylation reactions. Imidazole-functionalized copolymers of poly(methyl methacrylate-co-3-imidazolyl-2-hydroxy propyl methacrylate) were used for intramolecular Cu-imidazole binding that triggered the self-folding of polymers. Polymer chains imposed steric hindrance which yielded unsaturated Cu sites with an average coordination number of 3.3. Cu-containing SCPNs showed a high selectivity for the hydroxylation reaction of phenol to catechol, >80%, with a turnover frequency of >870 h-1 at 60 °C. The selectivity was largely influenced by the flexibility of the folded polymer backbone where a more flexible polymer backbone allows the cooperative catalysis of two Cu sites. The second coordination sphere provided by the folded polymer that has been less studied is therefore critical in the design of active mimics of metalloenzymes.