Journal of Molecular Structure 1255 (2022) 132419

Contents lists available at ScienceDirect

Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Emerging strategies for microbial screening of novel

chemotherapeutics
Nidhi Srivastava a,b, Indira P. Sarethy b, Jaison Jeevanandam c, Michael Danquah d,∗
a
Research and Development Department, Decode DNA Pvt. Ltd., 16/2 Ashok Nagar, New Delhi, 110018A, India
b
Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, Noida, 201309, India
c
CQM-Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus da Penteada, 9020-105 Funchal, Portugal
d
Department of Chemical Engineering, University of Tennessee, Chattanooga, TN 37403, United States

a r t i c l e i n f o a b s t r a c t

Article history: Enhanced antimicrobial drug resistance, virulence, pathogenicity, contagiousness, and lifestyle-associated
Received 10 September 2021 factors have led to various health-related syndromes. These syndromes require the urgent intervention
Revised 24 December 2021
of new chemotherapeutic agents as it can lead to significant mortality among different age groups of
Accepted 12 January 2022
the global population. Recent advancements in the screening methodologies for microbial culture selec-
Available online 13 January 2022
tion have contributed considerably to the field of drug development, since microbial sources continue to
Keywords: provide the best skeletons for natural product-based drugs. Several culture-based conventional microbial
Microbial screening screening and high-throughput metagenomic sequencing approaches have provided the platform for the
Chemotherapeutics discovery of numerous novel microbes and their products from various niche habitats. However, vari-
Metabolite fingerprinting ous underexplored habitats required further investigation for the identification of novel microbial- and
Antibiotics chemo-diversity and to discover new drug molecules. New bioprospection strategies, such as harvest-
ing of products from rare microbes, have also contributed to the field of new drug discovery programs.
Similarly, advancements in molecular metabolite fingerprinting techniques associated with databases and
bioinformatic pipelines and software have played a pivotal role in microbial screening for drug discov-
ery. This article provides an overview of various screening approaches to identify new microorganisms,
metabolite fingerprinting techniques, and novel chemotherapeutics derived from microbial sources. Ad-
ditionally, recent computational approaches for high-throughput screening of microbes to discover novel
chemotherapeutics are discussed.
© 2022 Elsevier B.V. All rights reserved.

1. Introduction
The continuous widespread and evolution of multi-drug resis-
tant and the introduction of pan-resistant pathogens have neces-
sitated the exploration of novel drug molecules to combat them.
Various research studies have reported that patients with sec-
ondary infections caused by antimicrobial or multi-drug resis-
tant microbes suffer critical ailments compared to patients in-
fected with non-resistant microbes [1,2]. It is challenging for the
conventional antimicrobial agents to inhibit multi-drug resistant
pathogenic microbes. Hence, there is a significant demand for new
antimicrobial drugs. Drug discovery is a complex process, which
approximately takes 10–15 years to reach the final commercial
product and must adhere to astringent regulatory approval pro-
cess [3,4]. In the last few decades, drug discovery by combinato-
∗
Corresponding author.
E-mail address: michael-danquah@utc.edu (M. Danquah).
rial chemistry has resulted in several challenges, and this has re-
vived research interest into natural products for harvesting drug
molecules. It is estimated that a total of 1030 microbial cells ex-
ist on earth, with 106 –108 separate prokaryotic species [5,6]. It
is well-known that unique microbial and genetic diversity is con-
served among prokaryotes with a great potential for natural prod-
uct development due to a variety of in-built biosynthetic path-
ways [7]. However, only 1% of microbial diversity has been ex-
plored for novel chemotherapeutics that are beneficial for the
treatment of pathogenic infections through microbial growth inhi-
bition [8]. The bioprospection of under-explored habitats provides
a good platform to utilize novel microbial diversity with unique
metabolic pathways and gene clusters to produce new bioactive
compounds, including biochemicals with antimicrobial efficacy [9].
Prokaryotes have been reported to produce various chemother-
apeutic molecules with antibacterial [10], antiviral [11], antifun-
gal [12], anti-inflammatory [13], antioxidant [14], and anticancer
activities [15].

https://doi.org/10.1016/j.molstruc.2022.132419
0022-2860/© 2022 Elsevier B.V. All rights reserved.
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
Recent advancements in the field of biotechnology and bioin-
formatics have accelerated drug discovery programs [16]. An expo-
nential increase in culture independent metagenomic, meta tran-
scriptomic, and meta-proteomic experimental results have helped
in exploring the hidden microbial world with functional di-
versity compared to conventional culture-based methods [17].
Genome mining of various niche habitats and whole genome se-
quencing of single cultured bacterium via high-throughput se-
quencing platforms has unraveled various biosynthetic gene clus-
ters, such as non-ribosomal peptide synthetase (NRPS), Polyke-
tide synthase (PKS), and NRPS/PKS hybrid to produce novel
antibiotics [18]. Similarly, parallel advancements in the pub-
lic databases [National Center for Biotechnology Information
(NCBI)/WILEY/National Institute for Interdisciplinary Science and
Technology (NIIST)/PUBChem/PubMed] and bioinformatic pipelines
and software [Natural Product Domain seeker (NaPDoS)/Antibiotics
and Secondary Metabolite Analysis Shell(anti-SMASH)/ Paired End
Assembler Sequence (PANDAseq)/Quantitative Insights into Micro-
bial Ecology (QIIME)] have contributed in the same direction [19].
Metabolite fingerprinting approaches based on chromato-
graphic and non-chromatographic techniques, such as Gas
Chromatography-Mass Spectroscopy, liquid chromatography-
mass spectroscopy (LC-MS) and matrix-assisted laser desorption
and ionization-time of flight with advantages in the instrument
technology and regular updates of associated databases, create op-
portunities to familiarize the type of molecules a host can possess
or able to secrete [20]. Improvisations structure elucidating tech-
niques, such as Nuclear Magnetic Resonance, X-ray crystallography
and Raman Spectroscopy, have also depicted a clear picture of
biomolecules in multidimensional facets and enabled researchers
to identify the functional attributes of specific biomolecules in
metabolic processes and signal transduction pathways [21]. This
article presents an overview of various screening approaches to
identify new microorganisms, metabolite fingerprinting techniques,
and novel chemotherapeutics derived from microbial sources. In
addition, the article discusses bacteria and fungi that have been
reported to produce significant chemotherapeutic drug molecules
with an emphasis on screening techniques used in the identi-
fication of novel microorganisms and evolving technologies in
fingerprinting of metabolites via metabolomics for the discovery
of new drug molecules from microbes.
2. Advances in approaches to screen novel microorganisms
There are basically two approaches (conventional culture-
based and culture independent methods) for extracting the novel
chemotherapeutic agents from hidden microbial diversity and from
under-explored microbial consortia.
2.1. Selection of underexplored habitats
In biosphere, microbes are the essential elements of ecolog-
ical dynamics with more diffused life forms in variety of sub-
strates or niche habitats and endowed with diverse sources of nat-
ural bioactive molecules. However, majority of their structural and
functional diversity is not known. To develop novel chemothera-
peutic agents, under-explored habitats is the best choice to avoid
discovery of already explored molecules at high cost. Many stud-
ies have substantiated that under-explored areas have contributed
to assess novel bio- and chemo-diversity. Recently, a novel Strep-
tomyces variabilis RD-5 has been obtained from Gulf of Khambhat
(sea sediment), Gujarat in India, and exhibits good antioxidant and
antimicrobial activities [22]. Similarly, Paenibacillus sambharens is
that shows good antimicrobial activity has been isolated from halo-
alkaline lake from Rajasthan in India [23]. Many novel antimicro-
bial Kitasatospora sp., Actinomadura and Streptomyces have been ex-
2
Journal of Molecular Structure 1255 (2022) 132419
tracted from under-explored Timli forest in India [24]; Pseudono-
cardiaceae sp. TD-015 [25] and Yuhushiella sp. TD-032 sp. from
Thar desert in India [26]; an endophyte Trichoderma harzianum X-
5 from marine alga Laminaria japonica(marine alga), obtained from
Chang islands in China [27]. Nocardiopsis valliformis OT1, an alka-
liphilic actinobacterium exhibiting cytotoxic and antibacterial ac-
tivity has been obtained from Lonar crater in India [28]. Moreover,
novel bacteria (Phormidium sp., Cyanobacterium sp.) exhibiting anti-
cancer and antilarval activities have been obtained from hot spring
in Lesvos, Greece [29].
2.2. Conventional culture-based approaches
Culture-based methods utilize pure microbial cultures to iden-
tify their taxonomic novelty and to extract novel bioactive com-
pounds. However, only about 1% of microbial community has been
investigated using such conventional approaches. A major lim-
itation of this approach is the requirement of complex or se-
lective/enrichment medium with optimum growth conditions re-
quired by diverse microbes and challenges associated with main-
taining these in the laboratory [30]. However, advancements in
new technologies have increased capacity to explore novel isolates
by taxonomic delineation. Numerous modifications in basic me-
dia have made it possible to culture a variety of microbes that
are difficult to grow. La Scola et al. (2014)added antioxidants (glu-
tathione and ascorbic acid)to a base media to cultivate unculti-
vatable strict anaerobes from human gut [31]. Some of the un-
cultivable firmicutes (Peptoniphilus timonensis, P. grossensis and P.
obesiensis, Kallipygamas siliensis), bacteriodetes (Alistipes obesiensis)
and actinobacteria (Timonella senegalensis) have been cultured us-
ing rumen fluid from the human gut [32]. Likewise, Datta et al.
(2011) have used carrageenan as a gelling agent, rather than agar,
to investigate extreme alkaliphiles from soda ash effluents. Sim-
ilarly, the culture-based techniques have been modified and im-
provised to enhance the selective activity (antimicrobial, antifun-
gal and others) of the microbes [33]. Sa-uth et al. (2018)used a
modified tryptone soy broth with altered carbon and nitrogen to
enhance the antifungal activity of XenorhabdusstockiaePB09 against
plant pathogens (Fusarium oxysporum, Phytophthora sp., Pythium sp.
and Rhizoctonia solani) [34]. In addition, Zhang et al. (2020) used
modified M9 medium with the addition of sucrose, ammonium
chloride, and magnesium sulfate to enhance the antibacterial ac-
tivities of Bacillus amyloliquefaciens-9 isolated from the intestine of
Chiloscyllium plagiosum (bamboo shark) [35].
2.3. Culture independent methods
Culture-based methods have high discovery rates for well-
known compounds. High-throughput screening methods and/or
culture independent methods, such as metagenomic approaches
employing heterologous host expression to screen novel bioactive
molecules directly from the environment within a short duration
and with precision, have become a focus for research. These ap-
proaches have the potential to explore and exploit vast bio- and
chemo-diversity. If we consider soil microbial diversity, approxi-
mately thousands of new species can be present in one gram of
soil sample; however, only 1% is cultivable in laboratory conditions
[36] and the rest require exploration. High-throughput next gen-
eration sequencing platforms, such as Illumina, Ion Torrent, Pacific
Biosciences Real Time Sequencer, and Roche 454 (FS FLX+, GS Ju-
nior) possess ability to provide pieces of genome cluster informa-
tion in a large puzzle that require assembly to manifest compre-
hensive details about structural and functional diversity [37]. The
advantages of these techniques include the use of small sample
quantities, rapid processing (sequencing),inexpensive process, and
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
high precision [38]. These techniques are now emerging as mon-
umental advances in the field of molecular taxonomy and facili-
tating the investigation of novel biosynthetic gene clusters in the
hidden microbial world that can be utilized as lead molecules.
These approaches are currently revolutionizing studies on glob-
ally emergent challenges, such as pandemics, that are caused by
novel microbes or drug resistant microbes [39]. Functional and de-
scriptive metagenomics, metabolomics, meta-transcriptomics, and
meta-proteomics are the main types of culture independent meth-
ods for screening microbial chemotherapeutics.
2.3.1. Functional metagenomics
Functional metagenomics is based on the functional activity of
biosynthetic pathways without culturing of the microbes. A broad
spectrum antibiotic named Turbomycin A and B, exhibiting broad
spectrum activities against gram positive and negative bacteria
were developed using this approach [40]. For the assessment of
novel antimicrobial activities of microbes from selected niche habi-
tats, two approaches are generally followed; (i) direct searching of
clones that can inhibit pathogens or (ii) identification of polyke-
tide sequences and synthetases that constitute a relevant family of
drugs [41].
2.3.2. Descriptive metagenomics
Descriptive metagenomics describe the distribution of microbial
species by sequencing 16S rDNA genes, thereby providing details of
the microbial dynamics of the microbes in any ecosystem. It also
facilitates the understanding of global metabolism in the ecosys-
tem [41]. This correlated information helps to understand the phar-
macodynamics and pharmacokinetics of the secreted molecules.
Initially, the microbial population analysis is centered on ribosomal
RNA analysis. The denaturing gradient and sequencing via gel elec-
trophoresis is then applied to describe the abundant species. How-
ever, high-throughput methodologies have now enabled a more
comprehensive and precise approach. More in-depth species dis-
tribution patterns are analyzed by 16S RNA microarrays and next
generation sequencing methods [42]. These methods also allow the
establishment of correlations between the microbiota and associ-
ated disorders, level of antibiotic resistance, and associated dis-
eases. Metagenomic approaches have also favored the investiga-
tion and detection of certain significant gene clusters related to
drug discovery, such as Polyketide Synthases (PKSs) (I, II, III), Non-
ribosomal Peptide Synthetase (NRPS), PKSs and NRPS hybrid and
Post-Translationally Modified Peptides (RiPPs).
Basically, two approaches, namely random sequencing, and tar-
geted sequencing, are used in metagenomics. In random sequenc-
ing, initially high-quality environmental DNA is extracted and frag-
mented randomly for size analysis via gel electrophoresis. Later,
a library is prepared, sequencing is performed at high through-
put, and the sequences are assembled. This method is applied
to genome, metabolic processes, and metabolite characterization
of bacteria, archaea, and viruses [43]. Targeted metagenomic se-
quencing covers genetic composition and structure required in
determining the bioactive potential of genes or gene clusters
by evaluating the differences and homology between standard
and target gene/gene clusters [44]. Application of more advanced
screening and amplification methods, such as Multiple Displace-
ment Amplification (MDA), Denaturing Gradient Gel Electrophore-
sis (DGGE), Whole-Genome Amplification (WGA), in combination
with metagenomics has strengthened the detection approach. Sim-
ilarly, integrated study of PCR, mutagenesis, and chimeragene-
sis with metagenomics has favored the investigation of func-
tional genes. The mRNA recovery and novel gene detection from
metagenome have been successful due to the inclusion of vari-
ous techniques, such as Fluorescence-Activated Cell Sorting (FACS),
Community Isotope Array (CI Array), Phenotypic Micro-array (PM).
3
Journal of Molecular Structure 1255 (2022) 132419
SIGEX (Substrate-Induced Gene Expression) and METREX (Metabo-
lite Regulated Expression)are recently developed methods that
have promoted more effective functional analysis [45].
2.3.3. Metabolomics
Metabolomics is an emerging field in biotechnology that cov-
ers the identification and quantification of metabolites in complex
biological systems, giving a comprehensive picture of novel drug
scaffolds, functional activity, drug targets and associated binding
patterns in a high throughput manner for drug discovery [46,47].
It is of more interest to identify robust microbiome drug targets by
investigating gene consortia and their products impacting human
diseases along with microbial diversity, rather than only focus-
ing on the identification of microbial taxa. Annotations of microbe
metabolite secretome are more significant than other approaches
for analyzing disease biomarkers and drug targets due to the sec-
retary biomolecules such as antimicrobials, probiotics, toxins, small
peptides, short chain fatty acids, bile acids, urea, and glycerol. that
can easily cross the epithelial barriers of the host than the mi-
crobe itself. The annotation is coupled with basically Mass Spec-
trometry (MS) or Nuclear Magnetic Resonance (NMR) based tech-
niques to identify metabolites in germ free conditions. The tech-
nique is highly robust; however, the study reported by Johnson
et al. (2015) and da Silva et al. (2015), demonstrated that less
than 2% of data generated by the spectroscopy and other methods
could be annotated due to unavailability of metabolite information
in databases [48,49]. Nevertheless, the molecular networking such
as Global Natural Products Social molecular networking (GNPS)
for molecular annotations of mass spectra has been strengthened
with an accompanying 41% increase in unique compound matches,
144% increase in total matched spectra in public database, 767%
increase in public and private data in the libraries with 4% overall
increase in library size [50]. Subsequent implementation of various
methods such as Bayes and target-decoy based methods [51] and
feature-Based Molecular Networking (FBMN) have also strength-
ened GNPS for the detection and annotation in Mass Spectrome-
try [52]. Many reports have been published to describe the role of
metabolomic study in detecting and curing diseases. For instance,
Hsiao et al. (2013) conducted a metabolomics study and identi-
fied that this technique is suitable to probe and treat neuro de-
velopmental disorders, including autism spectrum disorder (ASD)
by probiotic treatment. They demonstrated that affected children
had increased intestinal permeability due to increased expression
of the gene CLDN15 and decreased gene expression of TJP1, TJP2,
OCLN, and CLDN8. Gut permeability has been corrected by oral
treatment of human commensal Bacteroides fragilis and also re-
solved behavioral abnormalities [53]. Recently, MS has been found
to be a suitable technique for the identification of ASD such as
fragile X syndrome (FXS) and Smith-Lemli-Opitz Syndrome (SLOS)
by analyzing protein change endogenously [54].
2.3.4. Meta-transcriptomics
Meta-transcriptomics allow gene expression analysis within
natural niche habitats, providing information about the functional
biodiversity of the molecules. The technique has also been use-
ful in screening and analyzing pathogens, antimicrobial produc-
ers, antibiotics resistomes, role of xenobiotics, and other molecules.
More advanced and combined analysis of metagenomics and meta-
transcriptomics of biological samples has opened new avenues to
understand lesser-known concepts about microbial ecology as well
as dynamics and have served a valuable purpose in elucidating
drug discovery pathways [55]. Meta-transcriptomic analysis from
microbiomes involves the total RNA. Only mRNA is selected as it
is directly involved in the gene expression. The mRNA is subse-
quently fractionated, cDNA library is prepared and ligated with
the adaptors, which is amplified, and finally sequenced to generate
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
millions of RNA-sequence reads that are mapped with reference
genome to analyze the expressed genes. Further, high-throughput
and versatile techniques are required to derive specific findings
from increasing the size and number of datasets that are produced
from this technique. Several bioinformatics tools, such as Trimmo-
matic55 for quality filtering, HUMAnN, and MG-RAST have been
developed for the annotation, CuffDuff for differential gene expres-
sion, BOWTIE and GEM for mapping purposes.
The meta-transcriptomics technique has been identified to be
very effective in drug discovery. For instance, a study by Haiser and
Turnbaugh (2012) showed that the technique can provide informa-
tion about drug efficacy. In their study, the gut microbiota tran-
scriptional profile exhibited that Eggerthella lenta had cytochrome-
encoding operon that was upregulated in the presence of the anti-
cancer drug digoxin and inactivate it. Increased dietary proteins re-
duced the metabolic degradation of digoxin and enhanced the con-
centration of the drug in the serum. However, several challenges,
such as skewed antibiotic resistance genes (ARGS) database and
inappropriate investigation of allelic ARGS variants due to lower
frequency, have limited the technology in drug discovery [56].
2.3.5. Metaproteomics
Metaproteomics provide deep proteomic insights of the nat-
ural niche habitats. Mass spectrometric-metaproteomic analyses
can characterize proteins quantitatively, predict the microbiome’s
abundance and functions. Microbial biomass is provided by pro-
teins; hence, by estimating the protein biomass by metaproteomic
read out, structural community can also be predicted. There are
several metaproteomic approaches that are applied in novel drug
discovery. For instance, Rapid Assay of Individual Microbiome
(RapidAIM) metaproteomic technique has been developed by Li
et al. (2019) to determine the microbiome response towards drug
compounds. They demonstrated taxon-specific biomass and func-
tional contributions, alterations in biomass, and response of en-
zymatic pathways in gut microbiota. They evaluated forty-three
compounds with different properties, such as antibiotics, antidia-
betic drugs, and antipsychotics, against five individual microbiomes
and identified that, apart from the antibiotics (Ciprofloxacin, Isoni-
azid, Metronidazole and Rifaximin), berberine and ibuprofen were
also inhibitory to biomass accumulation during in vitro growth
of the microbiota [57]. Along with the antibiotics, berberine and
ibuprofen, fructo-oligosaccharide (FOS) altered metaproteome glob-
ally with significant functional responses of the respective com-
pounds. Recently, Heravi et al. (2020) employed the same tech-
nique to reveal taxonomic profiling of bacterial transcripts and as-
sociated severity in diabetic foot infections (DFIs) in patients at
Liverpool Hospital in Australia. The data showed the presence of
14 phyla with differences in abundance of microbial population in
severe and mild infections [58]. Staphylococcus aureus and Porphy-
romonas asaccharolytica were abundant in severe infections. The
technique also revealed one hundred and thirty-two biosynthetic
metabolic pathways, 131 antibiotic resistance genes, iron acquisi-
tion systems, and virulence factors [58]. Major challenges associ-
ated with the technique, which include inadequacies of the pro-
tein identification with confidence due to enormous genetic het-
erogeneity with uneven species distribution and differential pro-
tein expression, requiring a homology with the reference protein
in existing databases, can be overcome by combined genomics,
transcriptomics, and meta-proteomic approaches known as multi-
proteomic-genomics [47,59]. Moreover, the technique has become
more useful in deciphering molecular changes in the vicinity and
molecular mechanisms behind the outcome. The technique has as-
sisted in the interpretation of multiple mutations (generally occur
in cancer), Single Nucleotide Polymorphism (SNPs) and associated
disease status by effective monitoring of resultant transcripts and
translated proteins [60].
4
Journal of Molecular Structure 1255 (2022) 132419
3. Advances in molecular methods for fingerprinting of
metabolites
Metabolite fingerprinting is a powerful tool to characterize
bioactive molecules from biological systems by employing vari-
ous techniques, such as Gas Chromatography-Mass Spectroscopy
(GCMS–), High Performance Liquid Chromatography (HPLC), Ul-
tra High Performance Liquid Chromatography (UHPLC), Liquid
Chromatography-Mass Spectroscopy (LC-MS) Mass-Spectroscopy
(MS), Nuclear Magnetic Resonance (NMR), and Matrix-Assisted
laser Desorption/Ionization- Time of Flight (MALDI-TOF), along
with other hyphenated techniques. The advancements in these
techniques have enabled researchers to explore the vast repertoire
of bioactive compounds from uncultivated microbes and silent
biosynthetic gene clusters that can directly participate in drug
discovery pipelines. Various approaches, such as chromatographic,
non-chromatographic, and structure-elucidating are used for fin-
gerprinting of metabolites.
3.1. Chromatographic, non-chromatographic and structure elucidating
methods
Although there is a massive development in screening, extrac-
tion, separation, and characterization methods for drug molecules
from natural resources, isolation of compounds with medicinal val-
ues is still challenging. Advantages of hybrid methodology in chro-
matography and non-chromatography-based methods have facili-
tated several recent drug screening programs. Liquid Chromatogra-
phy, Gas Chromatography, and Capillary Electrophoresis associated
with distinct non-chromatographic systems, such as Fourier Trans-
form Infrared Spectroscopy (FTIR), Photodiode-Array Detection
(PDA), NMR, MS, and their hybrid techniques (LC-FTIR/LC–NMR/LC-
MS/HPLC-PDA/HPLC-MS/GC–NMR/GC-FTIR) have contributed to the
detection of desired drug molecules in raw unprocessed samples as
shown in Fig. 1.
The details of all these techniques have been previously re-
ported [61]. Advanced high-throughput and regular data up-
dates using these methods have become a key feature in de-
veloping hitherto new molecular drug compounds in drug dis-
covery. Ajilogba and Babalola (2019) identified sixty-eight com-
pounds from Bacillus amyloliquefaciens, B. thuringiensis, and Bacil-
lus sp., from the rhizosphere soil of Barabara groundnut from
North-West University agricultural farm in South Africa via GC–
MS technology [62]. The study demonstrated that many of these
extracted molecules could have antibacterial activity against B.
cereus, P. aeruginosa and E. feacalis. Ghssein et al. (2016) used
advanced Hydrophilic Interaction Liquid Chromatography (HILIC)
with Electrospray ionization-Mass Spectroscopy (ESI-MS) to iden-
tify a siderophore compound (staphylopine), secreted by Staphy-
lococcus aureus, that are associated with its virulence as it se-
questers metals (Iron/copper/zinc) from host for its survival and
is transcribed by a specific conserved mettallophore operon. The
associated biosynthetic pathway could be beneficial for the dis-
covery of antimicrobial agents that can inhibit methicillin re-
sistant S. aureus (MRSA) [63]. Recently, Sugrani et al. (2020)
demonstrated that LC-MS/MS technique can be useful in identi-
fying two novel peptides [QA1d-4 (HAILRGLLCLSLTLAFQPAF) and
QA1d-8 (ALHLPLKLMLRPALPLRLKLTL)] with antimicrobial and cy-
totoxic activities from Vibrio sp. strain ES25 [64]. Nguyen et al.
(2020) used High Resolution-Quadruple-Time of Flight-Electrospray
Ionization-Mass Spectroscopy (HR-Q-TOF ESI/MS/MS) and 2D NMR
analyses to characterize lobophorin A analogs and a furan-type
unique molecule exhibiting antibacterial and anticancer activity
from Streptomyces sp. VN1 [65]. GC-FTIR provides high resolu-
tion and precise spectra using rotational and vibrational energy
states of the molecule [66]. The technique gives information about
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
Fig. 1. Chromatographic and non-chromatographic methods for screening, separation, and characterization of biomolecules in drug discovery.
the functional group(s) present in the compound and their ar-
rangements. Gas Chromatography/Direct Deposition Fourier Trans-
form Infrared (GC/DD-FTIR) spectroscopy also provides information
about the functional groups and C–C double bonds in a molecule
[67]. The technique GC/DD-FTIR has been used to structurally elu-
cidate Sulfinamide 19 drug candidate from Salinispora pacifica CNS-
863 [68]. De Cesare et al. (2020) have used MALDI-TOF, combined
with Deubiquitylating enzymes assay (DUB), to evaluate the poten-
tial of deubiquitylating enzymes as drug targets [69]. Similarly, Ku-
rita et al. (2015) applied Liquid Chromatography-High Resolution
Mass Spectrometry (LC–HRMS) to more than two hundred bacte-
rial isolates to identify natural products [70]. Some of the latest
technologies, such as whole-cell solid-state NMR with more selec-
tive isotopic labeling, have been used to characterize antimicrobial
peptides [71].
3.2. Other high throughput screening methods
Emerging multi-drug resistant pathogens have catalyzed the de-
velopment of efficient high-throughput techniques, that can effi-
ciently screen multiple lead molecules simultaneously in a small-
time duration. Recently, Itoh et al. (2019) have developed a high-
throughput strategy (tandem mass spectrometry-sequencing and
two microscale antimicrobial activity assays) to develop novel ana-
logues of lysocin E, which is a peptide antibiotic extracted from
Lysobacter sp. Out of various analogs, eighteen samples exhib-
ited antimicrobial activity, compared tolysocin E against MRSA,
with a unique mode of action (disruption of bacterial membrane
by menaquinone-dependent mechanism) [72]. A new highly ef-
fective antibody-antibiotic conjugate (vancomycin-anti- S. aureus
antibody-vancomycin) has been developed by Lehar et al. (2015),
using combinations of various technologies (ELISA, HPLC, LC-MS,
Mass Quadrupole Time-of-Flight - Q-TOF, LC/MS, Flow cytome-
5
Journal of Molecular Structure 1255 (2022) 132419
try), to inhibit S. aureus via proteolytic cleavage of the antibi-
otics in phagolysosomes [73]. Numerous high-throughput sequenc-
ing technologies, such as sequencing by PacBio RS II, have en-
abled the identification of several novel polyketide gene clus-
ters,such as versipelostatin (VST) from Streptomyces versipellis
4083-SVS6 (Hashimoto et al. 2015), and fluvirucin B2 (flv) from
Actinomadura fulvaindica ATCC 53,714 [74]. Apart from these tech-
niques, the advent of monoclonal antibodies (mAbs) has facilitated
the discovery of novel drugs. A total of seventy-nine therapeu-
tic Abs has been commercialized out of five hundred and sev-
enty mAb safter approval by United States Food and Drug Ad-
ministration (US FDA) and thirty out of seventy-nine have been
used in the treatment of cancers. Some of the recently commer-
cialized mAbsare Caplacizumab (brand name: Cablivi) developed
by Ablynx, Romosozumab (brand name: Evenity) by Amgen/UCB,
Risankizumab (brand name: Skyrizi) by Boehringer Ingelheim Phar-
maceuticals/AbbVie Inc., and Brolucizumab (brand name: Beovu)
by Novartis Pharmaceuticals Corporation. These mAbs, mostly de-
veloped using hybridoma technology, have been used to treat
acquired thrombotic thrombocytopenic purpura, osteoporosis in
postmenopausal women at increased risk of fracture, plaque psori-
asis and macular degeneration, respectively [74,75].
4. Advances in bioinformatics and databases
The existing bioinformatics software and those that are used in
the molecular discovery pipeline have contributed not only to the
identification of drug targets, refinement, and candidate screen-
ing, but also in predicting drug resistance and side effect char-
acterizations. Accumulation of biomolecules (DNA/RNA/proteins)
with the development of protein structure simulation and ho-
mology modeling with associated informational databases about
small biomolecules have supported protein docking and virtual
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
screening strategies in various drug discovery programs. Bioinfor-
matics has led to the emergence of several fields (metagenomics,
meta-transcriptomics, metabolomics, meta-proteomics, computa-
tional genomics, and molecular phylogenetics) and provided a
comprehensive description of the structural and functional features
of molecules in selected niche habitats. In drug discovery pro-
gram, high-throughput molecular data is compared between symp-
tomatic and healthy (control) population. The comparison is made
to study the connection between symptoms of the disease and its
respective factors (mutation/epigenetic/environmental) modulating
gene expressions, drug target identification, and refining drug tar-
gets to achieve satisfactory outcomes with minimum side effects
and to analyze environmental impacts as well as drug resistance
potential [76].
Genetic diseases are of emerging concern in recent times.
Genome sequencing of the patient has resolved various genetic
disorders through the application of several bioinformatics strate-
gies and tools to compare genome sequences of healthy individ-
uals. For instance, somatic mutations are common in cancer pa-
tients and viral or bacterial infections. The use of bioinformat-
ics to probe somatic mutations may focus on three targets (1)
whether the mutation has created a Missense gene, (2) whether
the mutation occurred in conserved sequences, or (3) whether the
mutation occurred in known signal, such as regulatory motif or
splice sites or at initiation and/or termination sites hampering to
transcription. The third target is facilitated by the regulatory mo-
tif databases, where numerous pipelines are used to scan regu-
latory motifs. Example, position weight matrix (PWM) and Gibbs
sampler to provide information about the position of mutations
in regulatory domain; and de novo motif discovery and software,
such as Data Analysis in Molecular Biology and Evolution (DAMBE)
to extract coding sequences, tRNAs/rRNAs/exons/introns/5 and 3
splice sites/upstream-downstream sequences in a high-throughput
approach, have been used to identify cancer-mediated somatic mu-
tations by providing an input of annotated sequences. Regulatory
motifs may act as response elements of nuclear receptors to gener-
ate specific signal transduction pathways. Hence, the identification
of regulatory motifs usually can lead to refinement of drug targets
[76].
Various bioinformatic tools have been identified as favorable for
mutation screening by candidate gene selection, known as Target-
Induced Local Lesions in Genome (TILLING), analysis. It requires
Web-based tools, relevant repositories of genomics, gene expres-
sion repositories, and gene ontology (GO) databases for complete
functional annotation of the data and to establish the correlation
between mutations as well as drug targets. For instance, the dele-
terious (loss-of-function) mutation can be observed and identified
by Codons Optimized to Detect Deleterious Lesion (CODDLE) (free
web-based software) and sorting intolerant from tolerant (SIFT)
software [77]. RNA-Seq obtained from transcriptomic data can be
studied to identify the differences in targeting, control, and suf-
ferers in terms of gene expressions, gene regulation, and alternate
splicing approach using more advanced software, such as A Biosta-
tistical tool for Transcriptomics Analysis (ABioTrans) [78]. Similarly,
various bioinformatic tools and software are employed to investi-
gate drug targets and phenotypic screening as well as using pro-
teomic data. Public databases, such as Protein Abundant Database
(PaxDB), NIST libraries, Spectra Searching Tool (SpectraST) libraries,
BiblioSpec libraries, and Global Proteome Machine database store
proteomic information from almost all model organisms and help
in assessing different gene expression via differential protein pro-
duction amongst control and diseased populations [79,80]. There
are certain limitations with protein abundance data as low molec-
ular weight proteins, transient peptides, membrane proteins (elic-
itor of signal transduction pathways) are hard to screen, purify,
and characterize. Proteomic data prediction using transcriptomics
6
Journal of Molecular Structure 1255 (2022) 132419
sometimes create discrepancies in mRNA and protein abundance,
due to differential mRNA transcriptional and protein degradation
efficiencies. Hence, more advanced technologies as well as ribo-
some profiling associated with transcriptomics data can provide
better information about protein production rate to explore new
protein-coding genes, which can act as drug targets and help to in-
vestigate cellular mechanisms in changing environments [76]. Var-
ious databases or public resources related to proteomics in drug
discovery are provided in Table 1.
National Center for Biotechnology Information (NCBI) is a
database with nucleotide and protein blast pipelines that can help
in establishing the relatedness (homology/non-homology) to the
reference data and facilitate structural biology studies. It can be
noted that various bioinformatic software have also served to de-
tect resistance status of the pathogens and drug targets. They help
in identifying pathogenicity islands, virulence factors and mech-
anisms and mutational status associated with diseases [76]. Sev-
eral bioinformatics software, such as Antibiotic Resistance Gene-
Annotation (ARG-ANNOT), Carbon distribution (CARd), Short Read
Sequence Typing Read 2 (SRST2), MEGARes, Gene finder, Antimi-
crobial Resistance Identification by Assembly (ARIBA), Kmer Resis-
tance, AMR Finder, and ResFinder have been developed to explore
antimicrobial resistance determinants [81]. The drug discovery re-
lated software, databases, and web services belonging to different
groups have extensively been compiled and maintained by Swiss
Institute of Bioinformatics, which is accessible at http://click2drug.
org/ (Fig. 2) and can be categorized into the distinct groups as
shown in Table 2.
5. Novel chemotherapeutic agents from microbial sources
The spread of antimicrobials and increase in multi-drug resis-
tance amongst pathogens through over-consumption of antibiotic
have promoted the need to explore the discovery of novel an-
timicrobial compounds. Microbial sources have been an immense
source for several novel antibiotics. Under-explored habitats with
novel and/or rare microbial taxa have facilitated the quest of dis-
covering new antibiotics. Recently, abierixin, epinigericin, nigericin,
and novel grisorixin methyl ester antibiotics have been discov-
ered from Streptomyces youssoufiensis (obtained from Chelia Moun-
tain in Algeria) using high performance liquid chromatography-
electrospray ionization mass spectrometry techniques (HPLC-ESI-
MS) [82]. Various recently developed antibacterial, antifungal, an-
tiviral, anticancer, antioxidant and anti-inflammatory compounds
from microbial sources are shown in the Supplementary Table
3.1–3.6.
6. Computational approaches for high-throughput microbial
chemotherapeutic screening
Several novel computational approaches have been developed
and employed for high-throughput screening of microbes with
chemotherapeutic activity. Cheminformatics, bioinformatics, and
semantic (knowledge)-based methods involve computational tech-
niques for screening and designing of drugs. Cheminformatics are
classified into quantitative structure activity relationship (QSAR)
analysis, molecular docking and dynamics, mutagenesis and library
construction, gene expression perturbation, and modeling protein
structure and function. It offers the possibility for mining and
linking high-throughput sequencing data to putative disease treat-
ments to be beneficial for semantic-based drug screening meth-
ods [102]. Recently, biomolecular simulations, such as quantum
mechanics and molecular mechanics, molecular docking, pharma-
cophore modeling, and de novo methods are widely used for nat-
ural product-based screening and designing of drugs [83]. Further,
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al. Journal of Molecular Structure 1255 (2022) 132419

Table 1
Proteomics related databases/software useful in drug discovery.

Databases/ software/web service URL Functions

Chemical European Molecular Biology https://www.ebi.ac.uk/chembl/ A database of chemical, genomic, and bioactivity data to develop new drugs from
Laboratory (ChEMBL) genomic information.
SwissSidechain https://www.swisssidechain.ch/ Structural and molecular mechanics database of hundreds of non-natural
amino-acid sidechains that can be used for in silico investigation of their insertion
into natural peptides or proteins.
University of California, San Francisco https://www.cgl.ucsf.edu/chimera/ Interactive visualization and analysis of molecular structures and related data,
Chimera (UCSF Chimera) including density maps, trajectories, and sequence alignments.
SwissBioisostere http://www.swissbioisostere.ch/ Information and access of molecular replacements, useful for compound
optimization in drug design.
SwissDock http://www.swissdock.ch/ Web service for predicting molecular interactions between a target protein and a
small molecule.

SWISS-MODEL https://swissmodel.expasy.org/ Fully automated protein structure homology-modeling server, accessible via Expasy
web server or from DeepView (Swiss Pdb-Viewer).
Proteomics Identifications Database http://www.ebi.ac.uk/pride/ A centralized, standards compliant, public data repository for mass spectrometry
(PRIDE) proteomics data, including protein and peptide identifications, corresponding
expression values, post-translational modifications and supporting mass spectra
evidence.
Global Proteome Machine Database http://gpmdb.thegpm.org/ Proteomics data analysis, reuse, and validation.
PeptideAtlas http://www.peptideatlas.org/ Provides a list and link to Spectrum Libraries built specifically for spectrum library
searching of tandem mass-spec data.
NIST Libraries of Peptide Tandem http://peptide.nist.gov/ NIST peptide libraries are comprehensive, annotated mass spectral reference
Mass Spectra collections from various organisms and proteins useful for the rapid matching and
identification of acquired MS/MS spectra.

Fig. 2. Drug discovery pipeline (in silico) (Figure adapted from https://click2drug.org/).

several in silico computational techniques are used for gene essen-
tiality prediction for the screening of microbes with chemother-
apeutic compounds. Transposon sequencing methods, homology
mapping models, protein network topology models, metabolic net-
work reconstruction and simulation models are some of the in-
silico approaches used for microbial screening [84]. Becerra and
Lahoz-Beltra (2020) reported novel in silico methods developed for
fitness step evaluation in evolutionary algorithms, especially for
the screening of beneficial microbes. In this study, artificial intel-
ligence mediated genetic algorithm analysis via evolutionary com-
puting approach demonstrated to be beneficial for the screening of
useful microbes to extract, design, and develop chemotherapeutic
drugs based on the expression of operons, plasmids, genetic pat-
tern, conjugation, and DNA [85].
Machine learning and deep learning are the latest trends in
computational approaches for screening chemotherapeutic com-
pounds from microbial strains. It relies on the development and
utilization of computational methods that can adapt and learn

7
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al. Journal of Molecular Structure 1255 (2022) 132419

Table 2
Drug discovery related software, databases, and web services maintained by Swiss Institute of Bioinformatics.

Bioinformatic pipeline Examples

Databases NCBI, ChEMBL, and Protein DataBank (PDB)

Chemical structure representations
Molecular modeling and simulation
Homology modeling
Binding site prediction
Docking
Screening for drug candidates
Drug target prediction
Ligand design
Binding free energy estimation
QSAR (Quantitative structure-activity
relationship)
ADME (Absorption, distribution,
metabolism, and excretion) toxicity
Computer-Aided Drug-Design Platform using PyMOL, ChemDraw, and Marvin Suite
PyRosetta, Visual Molecular Dynamics (VMD), VEGA ZZ, and BALLView
Swiss-PDB Viewer, DeepView, ProModel, and ProSide
IntFOLD, MED-SuMo, TRAPP, CAVER, sc-PDB, and CASTp
Database of Useful Decoys: Enhanced (DUD.E.), GPCR-Bench., Virtual Decoy Sets (VDS), and Glide Fragment Library
DockoMatic, Autodock Vina plugin for PyMOL, Pharmer, Catalyst, PharmaGist, SwissSimilarity, and Blaster
SuperPred, and HitPick,
GANDI, LUDI, BREED, SwissBioisostere, VAMMPIRE, sc-PDB-Frag, e-LEA3D, and eDesign
Hyde, X-score, NNScore, DSXONLINE, BAPPLserver, and BAPPL-Zserver
cQSAR, SeeSAR, clogP, MOLEdb, ChemDB/Datasets, Datasets from the Milano Chemometrics and QSAR Research Group
QikProp, VolSurf, GastroPlus, ALOGPS, OSIRIS Property Explorer, and SwissADME

Fig. 3. High throughput machine learning-based approach in combination with other methods for the screening of microbes. Reproduced with permission from [88], ©Else-
vier.

without user instruction using statistical models and algorithms nomic classification of RNA viruses. The results showed that CHEER
for the analysis and identification of patterns in data [86,87]. can be beneficial in the analysis of real and simulated sequencing
Leavell et al. (2020) demonstrated that high throughput screening data with a higher accuracy compared to conventional alignment-
of microbes with chemotherapeutic compounds and other poten- free and alignment-based taxonomic assignment tools [93], which
tial drugs is possible via machine learning approaches as shown will be useful in screening viruses with chemotherapeutic com-
in Fig. 3. In this study, it was proposed that machine learn- pounds. Thus, machine and deep learning approaches in combina-
ing, especially unsupervised techniques, along with opto-fluidics tion with other existing computational, spectroscopic, and micro-
and acoustic mist ionization mass spectroscopy, will be benefi- scopic methods hold promise for efficient and rapid screening of
cial for the screening of microbes [88]. Weis et al. (2020) re- microbes with novel chemotherapeutic compounds and attributes.
ported that machine learning approaches can be useful for the
identification of antimicrobial susceptibility of microbes with the
7. Conclusion
help of MALDI-TOF mass spectroscopy. Artificial neural networks,
support vector machines, quick classifiers, and genetic algorithms
Advances in bioprospecting strategies, high-throughput tech-
are the latest machine learning approaches for microbial screen-
niques for screening compounds, regular update of databases, and
ing [89]. Likewise, deep learning methods, which is an artifi-
bioinformatics resources, along with advances in metabolomics
cial neural network-based machine learning approach with mul-
technologies, have accelerated novel drug discovery in an effec-
tiple processing layers for progressive extraction of higher-level
tive, selective, and specific manner from complex microbial natu-
features from the datasets [90], can be implemented for high-
ral products. Harvesting of novel biodiversity from untapped niche
precision microbial screening. For example, a deep learning-based
habitats has led to a vast repertoire of lead molecules. The treat of
screening web server named ‘Deep Screening’ for the acceleration
microbial pathogens in causing diseases and the multi-drug resis-
of drug discovery by generating de novo libraries have been re-
tance of pathogens is expected to continue. Hence, rapid scaling up
ported [91]. Jo et al. (2017) utilized a holographic deep convolu-
of drug discovery programs will become significant, and the speed
tional neural network learning approach for rapid optical screen-
with which technology is advancing will facilitate effective discov-
ing of anthrax spores. In this method, holographic unlabeled liv-
ery programs.
ing cell images and unique representation deep learning capability
for label-free and rapid screening was used for the identification
of Bacillus anthracis spores. This is one of the pioneering studies Declaration of Competing Interest
to utilize a deep learning method for the screening of microbes
[92]. Recently, Shang and Sun (2021) developed a novel hierarchi- The authors declare that they have no known competing finan-
cal classification computational model with convolutional neural cial interests or personal relationships that could have appeared to
network-based deep learning approach, named CHEER, for taxo- influence the work reported in this paper.

8
N. Srivastava, I.P. Sarethy, J. Jeevanandam et al.
Acknowledgements
Author (Indira P. Sarethy) is thankful to Jaypee Institute of In-
formation Technology, Noida for providing the necessary facili-
ties. Nidhi Srivastava acknowledges Decode DNA Pvt. Ltd., New
Delhi, and the author (Dr. Jaison Jeevanandam) would like to
thank the support from FCT-Fundação para a Ciência e a Tecnolo-
gia (Base Fund UIDB/00674/2020, Portuguese Government Funds)
and ARDITI- Agência Regional para o Desenvolvimento da Investi-
gação Tecnologia e Inovação through the project M1420–01–0145-
FEDER-0 0 0 0 05-CQM+ (Madeira 14–20 Program). All other authors
acknowledge their respective departments and university for the
support.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2022.132419.
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