Abstract
With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena.
To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents.
To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration.
To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B.
Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics.
The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.
Keywords
DNA nanotechnology to engineer the next generation of nanocarriers for drug and biomolecules delivery
In the past few decades, the progress made in the field of structural DNA nanotechnology has enabled the design and synthesis of a variety of DNA-based materials and DNA-based nanoarchitectures in 1-, 2-, and 3-dimensions [
[1]
]. More recently, the emergence of the DNA origami technology has revolutionized the fields of DNA nanotechnology and nanotechnology in general by providing a new strategy for designing and assembling discrete DNA nanoparticles with unprecedented structural fidelity [- Ke Y
- Castro C
- Choi JH.
Structural DNA nanotechnology: artificial nanostructures for biomedical research.
Annu Rev Biomed Eng. 2018; 20 (Jun 4Epub 2018 Apr 4. PMID: 29618223): 375-401https://doi.org/10.1146/annurev-bioeng-062117-120904
[2]
]. DNA origami assembly relies on folding a long single strand of DNA (ssDNA) called the scaffold, into any arbitrary shape simply using multiple complementary oligonucleotides that can hybridize in different locations on the scaffold, and ‘fold’ it into the desired form. This method yields monodisperse nanoparticles with nearly 100% identical structure and composition, which is unique in the realm of nanotechnology. In addition to exceptional programmability, DNA origami also offers several advantages over traditional biomaterials used to assemble nanocarriers [[3]
]. DNA origami is inherently biocompatible, easy to synthetize, allows spatial addressability to enable patterning of organic and inorganic molecules with nanoscale precision, and can be easily modified with various chemical groups for further functionalization. These properties allow researchers to design multifunctional nanoparticles that can carry a variety of molecules and target and/or track moieties simultaneously [- Zhang Y
- Tu J
- Wang D
- Zhu H
- Maity SK
- Qu X
- Bogaert B
- Pei H
- Zhang H.
Programmable and multifunctional DNA-based materials for biomedical applications.
Adv Mater. 2018; 30 (JunEpub 2018 Feb 1. PMID: 29389041)e1703658https://doi.org/10.1002/adma.201703658
[4]
]. DNA origami is now used to deliver cargo such as proteins, peptides and aptamers, and drugs [- Hu Y
- Niemeyer CM.
From DNA nanotechnology to material systems engineering.
Adv Mater. 2019; 31 (JunEpub 2019 Feb 15. PMID: 30767279)e1806294https://doi.org/10.1002/adma.201806294
[5]
,- Jahanban-Esfahlan R
- Seidi K
- Jahanban-Esfahlan A
- Jaymand M
- Alizadeh E
- Majdi H
- Najjar R
- Javaheri T
- Zare P.
Static DNA nanostructures for cancer theranostics: recent progress in design and applications.
Nanotechnol Sci Appl. 2019; 12 (Oct 15PMID: 31686793; PMCID: PMC6800557): 25-46https://doi.org/10.2147/NSA.S227193
[6]
] for many biomedical applications that require precision assembly of nanocarriers, such as targeted drug and nucleic acid delivery, immunotherapy and vaccine development [- Zhao S
- Tian R
- Wu J
- Liu S
- Wang Y
- Wen M
- Shang Y
- Liu Q
- Li Y
- Guo Y
- Wang Z
- Wang T
- Zhao Y
- Zhao H
- Cao H
- Su Y
- Sun J
- Jiang Q
- Ding B.
A DNA origami-based aptamer nanoarray for potent and reversible anticoagulation in hemodialysis.
Nat Commun. 2021; 12 (Jan 13PMID: 33441565; PMCID: PMC7807036): 358https://doi.org/10.1038/s41467-020-20638-7
[7]
]. However, multiple limiting factors (e.g., design complexity, structural characterization, and in vivo stability) have hindered the broader use of DNA origami in biomedical applications, despite its immense potential.- Zhao YX
- Shaw A
- Zeng X
- Benson E
- Nyström AM
- Högberg B.
DNA origami delivery system for cancer therapy with tunable release properties.
ACS Nano. 2012; 6 (Oct 23Epub 2012 Sep 13. PMID: 22950811): 8684-8691https://doi.org/10.1021/nn3022662
To solve some of these issues, Veneziano and colleagues have developed a complete set of methods to design and assemble 3D wireframe-based DNA origami with prescribed shape and size. They notably developed an algorithm named DAEDALUS that can automatically design DNA origami starting from a simple CAD design [
[8]
] to facilitate the tedious manual design process and enable shape and size matching for different applications. They also developed a simple and robust method for the synthesis of the DNA scaffold using an asymmetric polymerase chain (aPCR) reaction that has enabled production of pure scaffold with a prescribed sequence and length in a one-pot reaction [- Veneziano R
- Ratanalert S
- Zhang K
- Zhang F
- Yan H
- Chiu W
- Bathe M.
Designer nanoscale DNA assemblies programmed from the top down.
Science. 2016 Jun 24; 352 (Epub 2016 May 26. PMID: 27229143; PMCID: PMC5111087): 1534https://doi.org/10.1126/science.aaf4388
[9]
,- Bush J
- Singh S
- Vargas M
- Oktay E
- Hu CH
- Veneziano R.
Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.
Molecules. 2020; 25 (Jul 26PMID: 32722650; PMCID: PMC7435391): 3386https://doi.org/10.3390/molecules25153386
[10]
]. The team is now exploring methods to improve design of these nanoparticles for higher structural stability and more design flexibility, as well as finding new ways of accurately characterizing of the DNA origami nanoparticles produced [- Veneziano R
- Shepherd TR
- Ratanalert S
- Bellou L
- Tao C
- Bathe M.
In vitro synthesis of gene-length single-stranded DNA.
Sci Rep. 2018; 8 (Apr 25PMID: 29695837; PMCID: PMC5916881): 6548https://doi.org/10.1038/s41598-018-24677-5
[11]
]. These advances are critical to ensure reproducibility and facilitate scaling up production of these nanoparticles for biomedical applications.- Chiriboga M
- Green CM
- Hastman DA
- Mathur D
- Wei Q
- Díaz SA
- Medintz IL
- Veneziano R.
Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network.
Sci Rep. 2022; 12 (Mar 9PMID: 35264624; PMCID: PMC8907326): 3871https://doi.org/10.1038/s41598-022-07759-3
Furthermore, to assess the role of antigen nanoscale organization in activating immune cells and triggering a strong immune response, Veneziano and colleagues recently developed a versatile platform for immunogen delivery that offers complete control over the stoichiometry and nanoscale organization of antigens (i.e., proteins and peptides), and adjuvants. Using this strategy, they have demonstrated, in vitro, the importance of the organization and quantity of the eOD-GT8 HIV antigen to trigger B cell activation and, as such, have opened the way to exploring novel alternatives for rational vaccine development [
[12]
]. Leveraging these promising results, Veneziano and coworkers also applied this strategy to develop a DNA origami-based nanoparticle vaccine against SARS-CoV-2. Using the receptor-binding domain (RBD) of SARS-CoV-2 as the antigen, and multiplexing it with CpG adjuvants, they were able to demonstrate the efficacy of their platform to trigger immunity and provide protection against the virus in a hACE2 mouse model.- Veneziano R
- Moyer TJ
- Stone MB
- Wamhoff EC
- Read BJ
- Mukherjee S
- Shepherd TR
- Das J
- Schief WR
- Irvine DJ
- Bathe M.
Role of nanoscale antigen organization on B-cell activation probed using DNA origami.
Nat Nanotechnol. 2020; 15 (AugEpub 2020 Jun 29. PMID: 32601450; PMCID: PMC7415668): 716-723https://doi.org/10.1038/s41565-020-0719-0
Veneziano and colleagues are now leveraging their experience in construction and conjugation of DNA origami nanoparticles to explore novel methods aimed at controlling the temporal release of drug and other molecular cargoes. This will, in turn, enable the production of novel scaffolds for tissue engineering, and/or hydrogels for sustained release of drugs [
[13]
]. This new strategy relies on strand displacement and enables precise release of cargo molecules at various times to achieve desired physiological responses.- Hu CH
- Veneziano R.
Controlled release in hydrogels using DNA nanotechnology.
Biomedicines. 2022; 10 (Jan 19PMID: 35203423; PMCID: PMC8869372): 213https://doi.org/10.3390/biomedicines10020213
Lipid nanoparticles
The COVID-19 pandemic highlighted the significant potential of messenger RNA (mRNA)-based therapeutics as evidenced by the success of the Pfizer-BioNTech and Moderna vaccines. Advantages of using mRNA vaccines include cell-free manufacture of the mRNA, minimal risk of nucleic acid integration into the genome, and the ability to produce the target antigens from the vaccine mRNA in the cytoplasm. By packaging the mRNA in a lipid nanoparticle (LNP) delivery system, we can overcome mRNA's poor cellular uptake and its sensitivity toward enzymatic degradation [
[14]
]. The work at Mason focuses on the design of novel ionizable lipids for creating LNPs with improved properties for delivering mRNA.- Buschmann MD
- Carrasco MJ
- Alishetty S
- Paige M
- Alameh MG
- Weissman D.
Nanomaterial delivery systems for mRNA vaccines.
Vaccines (Basel). 2021; 9 (Jan 19PMID: 33478109; PMCID: PMC7836001): 65https://doi.org/10.3390/vaccines9010065
One of the key design parameters considered for optimal delivery efficiency is the pKa of the LNP as determined by 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) binding assay. Cullis and co-workers, who developed OnpattroTM, observed that high delivery efficiency was achieved when the pKa of the LNP was between 6 and 7 [
[15]
]. It was noticed that the theoretical pKa of novel lipids designed in silico were generally 2-3 units higher than the experimental pKa of the LNPs formulated from the same lipids. To experimentally determine the pKa of the ionizable headgroup of the lipids, Buschmann and colleagues synthesized water soluble analogues of the headgroups for NMR analysis at increasing pH values. As expected, the pKa of the headgroups matched the theoretically predicted pKa. A thermodynamic model was developed to explain the observed drop in pKa by accounting for the free energy of transfer of protons from the surrounding water to the lipid environment of the LNP and the electrostatic repulsion, or attraction of protons to the LNP. The effect is due to the fact that the pH is only measured in the aqueous phase, while the apparent pKa of the LNP that is measured by the TNS assay includes a partitioning of the protonated headgroup into the lipid environment of the LNP [[16]
].- Carrasco MJ
- Alishetty S
- Alameh MG
- Said H
- Wright L
- Paige M
- Soliman O
- Weissman D
- Cleveland TE
- Grishaev A
- Buschmann MD
Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.
Commun Biol. 2021; 4 (4th) (Aug 11PMID: 34381159; PMCID: PMC8358000): 956https://doi.org/10.1038/s42003-021-02441-2
Other factors that are considered in LNP design include the overall charge of the LNP as measured by the zeta potential and the shape of the aliphatic tail region of the lipids. The TNS assay measures the pKa at the surface of the LNP but does not report the overall charge of the LNP. Therefore, Carrasco and colleagues carried out electrophoretic mobility measurements to determine the zeta potential and thus the net charge of the LNP [
[16]
]. Consistent with the hypothesis that tissue specificity is influenced by charge, our approach enables designs to limit undesired systemic distribution by controlling the net charge of the LNP [- Carrasco MJ
- Alishetty S
- Alameh MG
- Said H
- Wright L
- Paige M
- Soliman O
- Weissman D
- Cleveland TE
- Grishaev A
- Buschmann MD
Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.
Commun Biol. 2021; 4 (4th) (Aug 11PMID: 34381159; PMCID: PMC8358000): 956https://doi.org/10.1038/s42003-021-02441-2
[17]
]. We also take into consideration the structure of the tail region of the lipids. A cone-shaped tail region can be formed by incorporating branched aliphatic chains, which are not compatible with lipid bilayer formation and may facilitate endosomal release [- Cheng Q
- Wei T
- Farbiak L
- Johnson LT
- Dilliard SA
- Siegwart DJ.
Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.
Nat Nanotechnol. 2020; 15 (AprEpub 2020 Apr 6. PMID: 32251383; PMCID: PMC7735425): 313-320https://doi.org/10.1038/s41565-020-0669-6
[18]
]. These parameters, among others tested in our laboratory, help to guide the design of lipids with improved physical properties for our formulated LNPs. Future directions of this work include developing the current technology for translation into the clinic, and expanding beyond vaccines to new therapeutic arenas.- Leung AK
- Tam YY
- Chen S
- Hafez IM
- Cullis PR.
Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems.
J Phys Chem B. 2015; 119 (Jul 16Epub 2015 Jul 7. PMID: 26087393): 8698-8706https://doi.org/10.1021/acs.jpcb.5b02891
Identification of protein-protein interaction regions
Protein–protein interactions are essential to the normal functioning of cells and modifications that alter these highly specific interactions can lead to cellular abnormalities and death. As such, identifying the contact points between proteins at the molecular level can open the door to a deeper understanding of the function of these complexes and the development of therapeutic interventions. Yet, determining the amino acid sequence of the contact sites between two proteins has been difficult. Thus, there has been a need for developing strategies that enable the direct analysis of sites of interaction between two native proteins in solution.
In 2014, Luchini and colleagues [
[19]
] used small-molecule dyes, such as Congo Red, to “paint” proteins and identify regions of protein-protein interaction. For the studies, they exposed preformed protein exposed to the molecular paints, resulting in high affinity noncovalent coating of the solvent-accessible surfaces of the complex (koff ≈ 10−5 s−1, whereas kon ≈ 102 m−1 s−1) [- Luchini A
- Espina V
- Liotta LA.
Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.
Nat Commun. 2014; 5 (Jul 22PMID: 25048602; PMCID: PMC4109009): 4413https://doi.org/10.1038/ncomms5413
[20]
]. This was followed by trypsin digestion, which the attached paint molecules inhibit, thus enabling the identification of the fragments of protein-protein interaction where the paint was excluded. The peptides that are present in the painted samples but absent from the controls, are considered candidates for protein-protein interaction regions. This technique is distinct from others such as hydrogen deuterium exchange and hydroxy radical labeling, because of the small molecule dyes employed and the native condition solution used. Once protein-protein interactions are identified using protein painting, the technique can be used to screen for molecules that can disrupt the interaction for therapeutic purposes [- Haymond A
- Dey D
- Carter R
- Dailing A
- Nara V
- Nara P
- Venkatayogi S
- Paige M
- Liotta L
- Luchini A.
Protein painting, an optimized MS-based technique, reveals functionally relevant interfaces of the PD-1/PD-L1 complex and the YAP2/ZO-1 complex.
J Biol Chem. 2019; 294 (Jul 19Epub 2019 Jun 5. PMID: 31167787; PMCID: PMC6643031): 11180-11198https://doi.org/10.1074/jbc.RA118.007310
[21]
].- Dailing A
- Mitchell K
- Vuong N
- Lee KH
- Joshi R
- Espina V
- Haymond Still A
- Gottschalk CJ
- Brown AM
- Paige M
- Liotta LA
- Luchini A
Characterization and Validation of Arg286 Residue of IL-1RAcP as a Potential Drug Target for Osteoarthritis.
Front Chem. 2021; 8 (Feb 3PMID: 33614593; PMCID: PMC7886681)601477https://doi.org/10.3389/fchem.2020.601477
To elucidate the interactions of among members of the interleukin 1 (IL-1) family of proteins, Günther and coworkers focused on the binding of interleukin-33 (IL-33) to its cognate receptor, soluble suppression of tumorgenicity (ST2), also known as sST2, IL-33R, IL-1R4, IL-1RL1 and T1. Upon binding of IL-33 to ST2, IL-1R accessory protein (IL-1RAcP) is recruited to the complex. Using protein painting, the authors have been able to identify differences in the way that IL-1 and IL-33 recruit IL-1RAcP and engage their respective receptors IL-R1 and ST2 [
[22]
]. Their studies revealed the presence of multiple interaction regions between IL-1RAcP and its receptor-cytokine partners, including the c2-d2 loop of IL-1RAcP that forms a loop binding both the cytokine and its receptor, Isoleucine (I) 155 of IL-1RAcP that acts as a hydrophobic hook, and the linker area between domains 2 and 3 of IL-1RAcP where binding to the receptor occurs. They also showed that the D3 domains of both receptors interacted.- Günther S
- Deredge D
- Bowers AL
- Luchini A
- Bonsor DA
- Beadenkopf R
- Liotta L
- Wintrode PL
- Sundberg EJ.
IL-1 Family Cytokines Use Distinct Molecular Mechanisms to Signal through Their Shared Co-receptor.
Immunity. 2017; 47 (Sep 19e4PMID: 28930661; PMCID: PMC5849085): 510-523https://doi.org/10.1016/j.immuni.2017.08.004
The dynamic picture that emerges from these studies suggests that the binding of the cytokine to its receptor needs to occur first to stabilize the D domains in a conformation that can bind IL-1RAcP. Binding of IL-1RAcP then follows. The next step is engagement of the receptor by hydrophobic hook around IL-1RAcPI155. The IL-1RAcP c2-d2 then binds the cytokine and primary receptor. This causes the coming together of the D3 domains.
The presence of loops that face the co-receptor binding site and can swap into IL-1β reducing the affinity of the binary complex for IL-1RAcP can explain the antagonistic activity of IL-1Ra. Yet, for IL-33, no cytokine antagonist has been identified that can block IL-33 signaling. In fact, for IL-33 regulation of signal transduction seems to occur through sST2, which is an alternatively spliced decoy receptor.
Thus, given that in contrast to IL-1R1, no antagonistic cytokine exists for ST2, and that IL-33 is its only ligand, it is plausible that IL-1RAcP has not needed to be regulated by IL-33 and that ST2 binding to it is sufficient. As such, Günther and colleagues suggest that IL-33 may not be a viable drug target and that its cognate receptor and co-receptors may be better therapeutic targets [
[21]
].- Dailing A
- Mitchell K
- Vuong N
- Lee KH
- Joshi R
- Espina V
- Haymond Still A
- Gottschalk CJ
- Brown AM
- Paige M
- Liotta LA
- Luchini A
Characterization and Validation of Arg286 Residue of IL-1RAcP as a Potential Drug Target for Osteoarthritis.
Front Chem. 2021; 8 (Feb 3PMID: 33614593; PMCID: PMC7886681)601477https://doi.org/10.3389/fchem.2020.601477
Antimicrobial peptide discovery through bioprospecting and other methods
The spread of antimicrobial resistance and the lack of new antibiotics presents an urgent threat to modern medicine. Antimicrobial peptides (AMPs) are essential elements of innate immunity in higher organisms and have been identified in nearly all organisms, including bacteria. AMPs include a large and diverse collection of peptides that demonstrate the ability to exert a direct antimicrobial effect against microbes, with many exerting broad-spectrum antimicrobial activities against bacteria, viruses, fungi and/or parasites. Today, there is a growing interest in AMPs as a potential resource for developing novel antibacterial agents to be used alone, or in conjunction with traditional antibiotics, against antimicrobial resistant (AMR), or multi-drug resistant (MDR) bacteria.
Van Hoek and colleagues have identified and characterized AMPs from mosquitoes [
[23]
] and bedbugs [- Kaushal A
- Gupta K
- Shah R
- van Hoek ML.
Antimicrobial activity of mosquito cecropin peptides against Francisella.
Dev Comp Immunol. 2016; 63 (OctEpub 2016 May 26. PMID: 27235883): 171-180https://doi.org/10.1016/j.dci.2016.05.018
[24]
]. Additionally, Van Hoek and Bishop have identified and studied an AMP from the Chinese king cobra [- Kaushal A
- Gupta K
- van Hoek ML.
Characterization of Cimex lectularius (bedbug) defensin peptide and its antimicrobial activity against human skin microflora.
Biochem Biophys Res Commun. 2016; 470 (Feb 19Epub 2016 Jan 21. PMID: 26802465): 955-960https://doi.org/10.1016/j.bbrc.2016.01.100
25
, - Amer LS
- Bishop BM
- van Hoek ML.
Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella.
Biochem Biophys Res Commun. 2010; 396 (May 28Epub 2010 Apr 23. PMID: 20399752): 246-251https://doi.org/10.1016/j.bbrc.2010.04.073
26
, - de Latour FA
- Amer LS
- Papanstasiou EA
- Bishop BM
- van Hoek ML.
Antimicrobial activity of the Naja atra cathelicidin and related small peptides.
Biochem Biophys Res Commun. 2010; 396 (Jun 11Epub 2010 May 10. PMID: 20438706): 825-830https://doi.org/10.1016/j.bbrc.2010.04.158
27
, - Dean SN
- Bishop BM
- van Hoek ML.
Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37.
Front Microbiol. 2011; 2 (Jul 4PMID: 21772832; PMCID: PMC3131519): 128https://doi.org/10.3389/fmicb.2011.00128
28
, - Dean SN
- Bishop BM
- van Hoek ML.
Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus.
BMC Microbiol. 2011; 11 (May 23PMID: 21605457; PMCID: PMC3397408): 114https://doi.org/10.1186/1471-2180-11-114
29
]. Furthermore, Bishop and van Hoek have experimentally identified novel AMPs from both the Komodo dragon and American alligator [- Blower RJ
- Barksdale SM
- van Hoek ML.
Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis.
PLoS Negl Trop Dis. 2015; 9 (Jul 21PMID: 26196513; PMCID: PMC4510350)e0003862https://doi.org/10.1371/journal.pntd.0003862
30
, - Bishop BM
- Juba ML
- Russo PS
- Devine M
- Barksdale SM
- Scott S
- Settlage R
- Michalak P
- Gupta K
- Vliet K
- Schnur JM
- van Hoek ML.
Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.
J Proteome Res. 2017; 16 (Apr 7Epub 2017 Feb 24. PMID: 28164707): 1470-1482https://doi.org/10.1021/acs.jproteome.6b00857
31
, - Bishop BM
- Juba ML
- Devine MC
- Barksdale SM
- Rodriguez CA
- Chung MC
- Russo PS
- Vliet KA
- Schnur JM
- van Hoek ML.
Bioprospecting the American alligator (Alligator mississippiensis) host defense peptidome.
PLoS One. 2015; 10 (Feb 11PMID: 25671663; PMCID: PMC4324634)e0117394https://doi.org/10.1371/journal.pone.0117394
32
]. A comprehensive list of AMPs can be found in the Antimicrobial Peptide Database (APD3) [- Juba ML
- Russo PS
- Devine M
- Barksdale S
- Rodriguez C
- Vliet KA
- Schnur JM
- van Hoek ML
- Bishop BM.
Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.
J Proteome Res. 2015; 14 (Oct 2Epub 2015 Sep 23. PMID: 26327436): 4282-4295https://doi.org/10.1021/acs.jproteome.5b00447
[33]
,- Wang G
- Li X
- Wang Z.
APD3: the antimicrobial peptide database as a tool for research and education.
Nucleic Acids Res. 2016; 44 (Jan 4Epub 2015 Nov 23. PMID: 26602694; PMCID: PMC4702905): D1087-D1093https://doi.org/10.1093/nar/gkv1278
[34]
]. Currently this database contains AMPs from six kingdoms of life (bacteria, archaea, protists, fungi, plants, and animals).- Wang G
- Zietz CM
- Mudgapalli A
- Wang S
- Wang Z.
The evolution of the antimicrobial peptide database over 18 years: Milestones and new features.
Protein Sci. 2022; 31 (JanEpub 2021 Sep 24. PMID: 34529321; PMCID: PMC8740828): 92-106https://doi.org/10.1002/pro.4185
To identify novel AMPs, Bishop and van Hoek developed a peptide discovery method that is independent of annotated genomes/genes and does not require the isolation and purification of potential peptides of interest. A central part of their BioProspecting discovery process utilizes custom-made hydrogel particles to preferentially enrich AMPs and AMP-like peptides from biological samples. The harvested peptides are then analyzed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS), using electron-transfer dissociation (ETD), in order to determine their sequences. Custom Python scripts have been developed to facilitate analysis of these peptide sequences for the identification of likely AMPs. This BioProspecting method is agnostic to the source of the peptides, and can be broadly used for the identification of potential peptides from biological samples prepared in aqueous buffers. Importantly, the BioProspecting particles can harvest peptides from small sample volumes, such as 100 microliters of plasma. This enables analysis and identification of peptides from rare and/or small animal species.
BioProspecting was employed in the identification of multiple novel peptides from the American alligator and the Komodo dragon [
30
, - Bishop BM
- Juba ML
- Russo PS
- Devine M
- Barksdale SM
- Scott S
- Settlage R
- Michalak P
- Gupta K
- Vliet K
- Schnur JM
- van Hoek ML.
Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.
J Proteome Res. 2017; 16 (Apr 7Epub 2017 Feb 24. PMID: 28164707): 1470-1482https://doi.org/10.1021/acs.jproteome.6b00857
31
, - Bishop BM
- Juba ML
- Devine MC
- Barksdale SM
- Rodriguez CA
- Chung MC
- Russo PS
- Vliet KA
- Schnur JM
- van Hoek ML.
Bioprospecting the American alligator (Alligator mississippiensis) host defense peptidome.
PLoS One. 2015; 10 (Feb 11PMID: 25671663; PMCID: PMC4324634)e0117394https://doi.org/10.1371/journal.pone.0117394
32
,- Juba ML
- Russo PS
- Devine M
- Barksdale S
- Rodriguez C
- Vliet KA
- Schnur JM
- van Hoek ML
- Bishop BM.
Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.
J Proteome Res. 2015; 14 (Oct 2Epub 2015 Sep 23. PMID: 26327436): 4282-4295https://doi.org/10.1021/acs.jproteome.5b00447
35
, - Barksdale SM
- Hrifko EJ
- Chung EM
- van Hoek ML.
Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii.
BMC Microbiol. 2016; 16 (Aug 19PMID: 27542832; PMCID: PMC4992317): 189https://doi.org/10.1186/s12866-016-0799-z
36
, - Barksdale SM
- Hrifko EJ
- van Hoek ML.
Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae.
Dev Comp Immunol. 2017; 70 (MayEpub 2017 Jan 13. PMID: 28089718): 135-144https://doi.org/10.1016/j.dci.2017.01.011
37
, - Chung EMC
- Dean SN
- Propst CN
- Bishop BM
- van Hoek ML.
Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.
NPJ Biofilms Microbiomes. 2017; 3 (Apr 11PMID: 28649410; PMCID: PMC5445593): 9https://doi.org/10.1038/s41522-017-0017-2
38
, - Hitt SJ
- Bishop BM
- van Hoek ML.
Komodo-dragon cathelicidin-inspired peptides are antibacterial against carbapenem-resistant Klebsiella pneumoniae.
J Med Microbiol. 2020; 69 (NovEpub 2020 Oct 21. PMID: 33084564): 1262-1272https://doi.org/10.1099/jmm.0.001260
39
]. These peptides demonstrated antibacterial activity against dangerous and multi-drug resistant bacterial pathogens. One of these peptides was developed into a new synthetic peptide called DRGN-1, which has shown antibiofilm, antibacterial and wound-healing capabilities against mixed-infected wounds in mice [- van Hoek ML
- Prickett MD
- Settlage RE
- Kang L
- Michalak P
- Vliet KA
- Bishop BM.
The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.
BMC Genomics. 2019; 20 (Aug 30PMID: 31470795; PMCID: PMC6716921): 684https://doi.org/10.1186/s12864-019-6029-y
[37]
,- Chung EMC
- Dean SN
- Propst CN
- Bishop BM
- van Hoek ML.
Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.
NPJ Biofilms Microbiomes. 2017; 3 (Apr 11PMID: 28649410; PMCID: PMC5445593): 9https://doi.org/10.1038/s41522-017-0017-2
[38]
]. The entire genome of the Komodo dragon was assembled by van Hoek and Bishop et al [- Hitt SJ
- Bishop BM
- van Hoek ML.
Komodo-dragon cathelicidin-inspired peptides are antibacterial against carbapenem-resistant Klebsiella pneumoniae.
J Med Microbiol. 2020; 69 (NovEpub 2020 Oct 21. PMID: 33084564): 1262-1272https://doi.org/10.1099/jmm.0.001260
[39]
] to assist in peptide identification via LC-MS/MS.- van Hoek ML
- Prickett MD
- Settlage RE
- Kang L
- Michalak P
- Vliet KA
- Bishop BM.
The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.
BMC Genomics. 2019; 20 (Aug 30PMID: 31470795; PMCID: PMC6716921): 684https://doi.org/10.1186/s12864-019-6029-y
In addition to the above approach, Van Hoek and collaborators have developed machine learning (ML) approaches to design novel antimicrobial peptides. They applied the Database-Filtering Technology (DFT) method to a set of peptides with activity against gram-negative bacteria, to select and synthesize eight novel synthetic antimicrobial peptides, three of which were active against gram-negatives, and one which was specific for gram-negative bacteria and did not kill gram-positive bacteria [
[40]
]. A recent review by Wang, Vaisman and van Hoek discusses machine learning tools such as cytotoxicity prediction, hemolytic prediction as well as additional antibacterial and antibiofilm activity computational predictors that are also being developed by researchers in the field [- Bobde SS
- Alsaab FM
- Wang G
- Van Hoek ML.
Ab initio designed antimicrobial peptides against gram-negative bacteria.
Front Microbiol. 2021; 12 (Nov 16PMID: 34867843; PMCID: PMC8636942)715246https://doi.org/10.3389/fmicb.2021.715246
[41]
].- Wang G
- Vaisman II
- van Hoek ML.
Machine learning prediction of antimicrobial peptides.
Methods Mol Biol. 2022; 2405 (PMID: 35298806; PMCID: PMC9126312): 1-37https://doi.org/10.1007/978-1-0716-1855-4_1
Van Hoek and colleagues are also searching for small molecules with the ability to alter bacterial virulence and pathogenic activity. A screen of FDA-approved drugs identified two polycyclic antidepressants, maprotiline and chlorpromazine, as having antibiofilm and anti-virulence activities against a biothreat model bacterium, Francisella novicida [
[42]
].- Dean SN
- van Hoek ML.
Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase Dependent Activity in Francisella novicida.
Virulence. 2015; 6 (PMID: 26155740; PMCID: PMC4601499): 487-503https://doi.org/10.1080/21505594.2015.1046029
In addition, diffusible signal factors (DSFs), long chain, cis-unsaturated fatty acids, have been identified by Dean, Chung and van Hoek as being the first known signaling molecule in Francisella [
[43]
]. These small molecules represent potential starting points for the development of adjunctive treatments that might accompany antibiotic treatment, especially in the case of drug-resistant bacteria.- Dean SN
- Chung MC
- van Hoek ML.
Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.
Appl Environ Microbiol. 2015; 81 (OctEpub 2015 Jul 31. PMID: 26231649; PMCID: PMC4579433): 7057-7066https://doi.org/10.1128/AEM.02165-15
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding
W81XWH2010054 DOD/United States
P41 GM103832/GM/NIGMS NIH HHS/United States
NSF EAGER (CCF-1547999)
K99EB030013 NIBIB NIH/HHS/United States
U. S. Army Research Office through the Institute for Soldier Nanotechnologies at MIT (Cooperative Agreement Number W911NF-18-2-0048)
R21-EB026008, R01-MH112694, AI048240, UM1AI144462, and UM1AI100663 NIH/HHS/United States
R01-AI 143740, R01-AI 146581 NIH/HHS/United States
1R21CA177535-01 NCI/NIH/HHS/United States
R01 AR068436/AR/NIAMS NIH HHS/United States
R21 CA177535/CA/NCI NIH HHS/United States
R33 CA173359/CA/NCI NIH HHS/United States
R33 CA206937/CA/NCI NIH HHS/United States
R01 AI132766/AI/NIAID NIH HHS/United States
HDTRA1-12-C-0039
DE-FC52-04NA25455 DOE/United States
R01AI105147 NIAID/ R01GM138552 GM/NIH/HHS/United StatesNIH/HHS/United States
DHS 2010-ST-061-AG0002
1R33CA173359-01 NCI/NIH/HHS/United States
1R21AR061075-01 NIAMS/NIH/HHS/United States
P50 GM103297/GM/NIGMS NIH HHS/United States
ONR (grants N00014-17-1-2609; N00014-16-1-2181; N00014-16-1-2953)
Human Frontier Science Program (RGP0029/2014)
P41GM103832/GM/NIGMS NIH HHS/United States
P50GM103297/GM/NIGMS NIH HHS/United States
References
- Structural DNA nanotechnology: artificial nanostructures for biomedical research.Annu Rev Biomed Eng. 2018; 20 (Jun 4Epub 2018 Apr 4. PMID: 29618223): 375-401https://doi.org/10.1146/annurev-bioeng-062117-120904
- Folding DNA to create nanoscale shapes and patterns.Nature. 2006; 440 (Mar 16PMID: 16541064): 297-302https://doi.org/10.1038/nature04586
- Programmable and multifunctional DNA-based materials for biomedical applications.Adv Mater. 2018; 30 (JunEpub 2018 Feb 1. PMID: 29389041)e1703658https://doi.org/10.1002/adma.201703658
- From DNA nanotechnology to material systems engineering.Adv Mater. 2019; 31 (JunEpub 2019 Feb 15. PMID: 30767279)e1806294https://doi.org/10.1002/adma.201806294
- Static DNA nanostructures for cancer theranostics: recent progress in design and applications.Nanotechnol Sci Appl. 2019; 12 (Oct 15PMID: 31686793; PMCID: PMC6800557): 25-46https://doi.org/10.2147/NSA.S227193
- A DNA origami-based aptamer nanoarray for potent and reversible anticoagulation in hemodialysis.Nat Commun. 2021; 12 (Jan 13PMID: 33441565; PMCID: PMC7807036): 358https://doi.org/10.1038/s41467-020-20638-7
- DNA origami delivery system for cancer therapy with tunable release properties.ACS Nano. 2012; 6 (Oct 23Epub 2012 Sep 13. PMID: 22950811): 8684-8691https://doi.org/10.1021/nn3022662
- Designer nanoscale DNA assemblies programmed from the top down.Science. 2016 Jun 24; 352 (Epub 2016 May 26. PMID: 27229143; PMCID: PMC5111087): 1534https://doi.org/10.1126/science.aaf4388
- Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.Molecules. 2020; 25 (Jul 26PMID: 32722650; PMCID: PMC7435391): 3386https://doi.org/10.3390/molecules25153386
- In vitro synthesis of gene-length single-stranded DNA.Sci Rep. 2018; 8 (Apr 25PMID: 29695837; PMCID: PMC5916881): 6548https://doi.org/10.1038/s41598-018-24677-5
- Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network.Sci Rep. 2022; 12 (Mar 9PMID: 35264624; PMCID: PMC8907326): 3871https://doi.org/10.1038/s41598-022-07759-3
- Role of nanoscale antigen organization on B-cell activation probed using DNA origami.Nat Nanotechnol. 2020; 15 (AugEpub 2020 Jun 29. PMID: 32601450; PMCID: PMC7415668): 716-723https://doi.org/10.1038/s41565-020-0719-0
- Controlled release in hydrogels using DNA nanotechnology.Biomedicines. 2022; 10 (Jan 19PMID: 35203423; PMCID: PMC8869372): 213https://doi.org/10.3390/biomedicines10020213
- Nanomaterial delivery systems for mRNA vaccines.Vaccines (Basel). 2021; 9 (Jan 19PMID: 33478109; PMCID: PMC7836001): 65https://doi.org/10.3390/vaccines9010065
- The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs.Nat Nanotechnol. 2019; 14 (DecPMID: 31802031): 1084-1087https://doi.org/10.1038/s41565-019-0591-y
- Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.Commun Biol. 2021; 4 (4th) (Aug 11PMID: 34381159; PMCID: PMC8358000): 956https://doi.org/10.1038/s42003-021-02441-2
- Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.Nat Nanotechnol. 2020; 15 (AprEpub 2020 Apr 6. PMID: 32251383; PMCID: PMC7735425): 313-320https://doi.org/10.1038/s41565-020-0669-6
- Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems.J Phys Chem B. 2015; 119 (Jul 16Epub 2015 Jul 7. PMID: 26087393): 8698-8706https://doi.org/10.1021/acs.jpcb.5b02891
- Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.Nat Commun. 2014; 5 (Jul 22PMID: 25048602; PMCID: PMC4109009): 4413https://doi.org/10.1038/ncomms5413
- Protein painting, an optimized MS-based technique, reveals functionally relevant interfaces of the PD-1/PD-L1 complex and the YAP2/ZO-1 complex.J Biol Chem. 2019; 294 (Jul 19Epub 2019 Jun 5. PMID: 31167787; PMCID: PMC6643031): 11180-11198https://doi.org/10.1074/jbc.RA118.007310
- Characterization and Validation of Arg286 Residue of IL-1RAcP as a Potential Drug Target for Osteoarthritis.Front Chem. 2021; 8 (Feb 3PMID: 33614593; PMCID: PMC7886681)601477https://doi.org/10.3389/fchem.2020.601477
- IL-1 Family Cytokines Use Distinct Molecular Mechanisms to Signal through Their Shared Co-receptor.Immunity. 2017; 47 (Sep 19e4PMID: 28930661; PMCID: PMC5849085): 510-523https://doi.org/10.1016/j.immuni.2017.08.004
- Antimicrobial activity of mosquito cecropin peptides against Francisella.Dev Comp Immunol. 2016; 63 (OctEpub 2016 May 26. PMID: 27235883): 171-180https://doi.org/10.1016/j.dci.2016.05.018
- Characterization of Cimex lectularius (bedbug) defensin peptide and its antimicrobial activity against human skin microflora.Biochem Biophys Res Commun. 2016; 470 (Feb 19Epub 2016 Jan 21. PMID: 26802465): 955-960https://doi.org/10.1016/j.bbrc.2016.01.100
- Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella.Biochem Biophys Res Commun. 2010; 396 (May 28Epub 2010 Apr 23. PMID: 20399752): 246-251https://doi.org/10.1016/j.bbrc.2010.04.073
- Antimicrobial activity of the Naja atra cathelicidin and related small peptides.Biochem Biophys Res Commun. 2010; 396 (Jun 11Epub 2010 May 10. PMID: 20438706): 825-830https://doi.org/10.1016/j.bbrc.2010.04.158
- Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37.Front Microbiol. 2011; 2 (Jul 4PMID: 21772832; PMCID: PMC3131519): 128https://doi.org/10.3389/fmicb.2011.00128
- Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus.BMC Microbiol. 2011; 11 (May 23PMID: 21605457; PMCID: PMC3397408): 114https://doi.org/10.1186/1471-2180-11-114
- Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis.PLoS Negl Trop Dis. 2015; 9 (Jul 21PMID: 26196513; PMCID: PMC4510350)e0003862https://doi.org/10.1371/journal.pntd.0003862
- Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.J Proteome Res. 2017; 16 (Apr 7Epub 2017 Feb 24. PMID: 28164707): 1470-1482https://doi.org/10.1021/acs.jproteome.6b00857
- Bioprospecting the American alligator (Alligator mississippiensis) host defense peptidome.PLoS One. 2015; 10 (Feb 11PMID: 25671663; PMCID: PMC4324634)e0117394https://doi.org/10.1371/journal.pone.0117394
- Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.J Proteome Res. 2015; 14 (Oct 2Epub 2015 Sep 23. PMID: 26327436): 4282-4295https://doi.org/10.1021/acs.jproteome.5b00447
- APD3: the antimicrobial peptide database as a tool for research and education.Nucleic Acids Res. 2016; 44 (Jan 4Epub 2015 Nov 23. PMID: 26602694; PMCID: PMC4702905): D1087-D1093https://doi.org/10.1093/nar/gkv1278
- The evolution of the antimicrobial peptide database over 18 years: Milestones and new features.Protein Sci. 2022; 31 (JanEpub 2021 Sep 24. PMID: 34529321; PMCID: PMC8740828): 92-106https://doi.org/10.1002/pro.4185
- Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii.BMC Microbiol. 2016; 16 (Aug 19PMID: 27542832; PMCID: PMC4992317): 189https://doi.org/10.1186/s12866-016-0799-z
- Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae.Dev Comp Immunol. 2017; 70 (MayEpub 2017 Jan 13. PMID: 28089718): 135-144https://doi.org/10.1016/j.dci.2017.01.011
- Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.NPJ Biofilms Microbiomes. 2017; 3 (Apr 11PMID: 28649410; PMCID: PMC5445593): 9https://doi.org/10.1038/s41522-017-0017-2
- Komodo-dragon cathelicidin-inspired peptides are antibacterial against carbapenem-resistant Klebsiella pneumoniae.J Med Microbiol. 2020; 69 (NovEpub 2020 Oct 21. PMID: 33084564): 1262-1272https://doi.org/10.1099/jmm.0.001260
- The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.BMC Genomics. 2019; 20 (Aug 30PMID: 31470795; PMCID: PMC6716921): 684https://doi.org/10.1186/s12864-019-6029-y
- Ab initio designed antimicrobial peptides against gram-negative bacteria.Front Microbiol. 2021; 12 (Nov 16PMID: 34867843; PMCID: PMC8636942)715246https://doi.org/10.3389/fmicb.2021.715246
- Machine learning prediction of antimicrobial peptides.Methods Mol Biol. 2022; 2405 (PMID: 35298806; PMCID: PMC9126312): 1-37https://doi.org/10.1007/978-1-0716-1855-4_1
- Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase Dependent Activity in Francisella novicida.Virulence. 2015; 6 (PMID: 26155740; PMCID: PMC4601499): 487-503https://doi.org/10.1080/21505594.2015.1046029
- Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.Appl Environ Microbiol. 2015; 81 (OctEpub 2015 Jul 31. PMID: 26231649; PMCID: PMC4579433): 7057-7066https://doi.org/10.1128/AEM.02165-15
Article info
Publication history
Published online: March 13, 2023
Accepted:
March 8,
2023
Received in revised form:
February 14,
2023
Received:
November 7,
2022
Publication stage
In Press Corrected ProofIdentification
Copyright
© 2023 The Authors. Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening.
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