Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS- CoV-2 activity

doi:10.21203/rs.3.rs-2706268/v1

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Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS- CoV-2 activity

https://doi.org/10.21203/rs.3.rs-2706268/v1

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The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly constrained peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used transcriptome mining, circular dichroism and mass spectrometric analysis to characterize fifteen new venom peptides. Some of these peptides were tested for their ability to bind to the SARS-CoV-2 spike protein and to inhibit the spike RBD - hACE2 interaction that precedes virus entry into the cell using a surface plasmon resonance-based assay. Seven peptides showed dose-dependent inhibitory effects. The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure of replication-competent SARS-CoV-2 to the synthetic peptide, we observed a two log10 PFU/mL reduction at sub-micromolar concentrations of the peptide compared to virus exposed to medium alone. Our results show that scorpion venom peptides could inhibit the SARS-CoV-2 spike RBD - hACE2 interaction, exhibit anti-SARS-CoV-2 activity through other unexplored modes of actions and represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides.

Biological sciences/Chemical biology

Biological sciences/Drug discovery

Physical sciences/Chemistry

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide (https://covid19.who.int/). Despite the accelerated development and deployment of vaccines, there is an urgent need for development of new therapeutics to combat the threat of new evolving SARS-CoV-2 variants against which current vaccines may be less effective. Currently approved small molecule antiviral drugs include remdesivir¹ and molnupiravir², which target the RNA-dependent RNA polymerase and nirmatrelvir which inhibits the main protease of the virus.³ As these targets are involved in viral replication, these drugs will only work after the virus infects the cell. Structural studies showed that the higher transmissibility of SARS-CoV-2 compared to previous coronaviruses is due to tighter binding of the viral spike glycoprotein to human angiotensin-converting enzyme 2 (hACE2).^{4, 5} This critical interaction precedes the viral entry into the cell and its inhibition is a key therapeutic target. Protein-protein interactions (PPI) are generally very challenging therapeutic targets for traditional small molecule drugs due to their highly dynamic and large interfacial surface.⁶ However, there is growing evidence that conformationally constrained and macrocyclic peptides can modulate these interactions and be valuable springboard for the design of effective small molecule PPI inhibitors.^{7, 8} Scorpion venoms are known to be rich in these conformationally constrained peptides of which several have been proven to possess antiviral activity against a wide range of viruses.^9–18 An example is the broad-spectrum antiviral venom peptide, Ev37 from the scorpion Euscorpiops validus that was found to be able to inhibit dengue virus type 2 (DENV-2), hepatitis C (HCV), Zika virus (ZIKV) and herpes simplex type I (HSV-1) in a dose dependent manner at noncytotoxic concentrations.¹⁴ Ev37 exhibits its action via alkalizing acidic organelles to prevent low pH-dependent fusion of the viral membrane-endosomal membrane, which mainly blocks the release of the viral genome from the endosome to the cytoplasm and then restricts viral late entry. SARS-CoV viruses are considered pH-sensitive viruses and their intracellular trafficking requires an acidic environment.¹⁹ Several other mechanisms have been reported for venom peptides and include direct virucidal effect, disturbance of the attachment of virus particles to the cell membrane surface, or interference with the virus replication.¹²

In this manuscript, we used mass spectrometry (MS) and venomous gland transcriptomic analysis to identify the sequence of new purified peptides from the venom of the north African scorpion Androctonus amoreuxi collected from Egypt. The chemistry of A. amoreuxi venom and its transcriptome have not been extensively studied as only 10 venom peptides have been identified and characterized to date (Table 1).^20–24 We have generated an annotated reference transcriptome for the A. amoreuxi venom gland. We also used a surface plasmon resonance-based assay to test the ability of the newly identified peptides to bind to the SARS-CoV-2 spike protein and to inhibit the spike RBD - hACE2 interaction. We synthesized the most active peptide and tested its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7).

Table 1

Peptides previously identified from *A. amoreuxi* venom
Peptide	Sequence	Ref
AamH1	VRDGYIVYPHNCVYHCIPSCDGLCKENGATSGSCGYIIKVGIACWCKDLPENVPIYDRSYKCY	20
AamH2	VRDGYIADAGNCGYTCVANDYCNTECTKNGAESGYCQWFGRYGNACWCIKLPDKVPIKVPGKCN	20
AamH3	ARDGYIAQPPNNCVYHCIPLSPGCDKLCRENGATSGKCSFLAGSGLACWCVALPDNVPIKIIGQKCT	20
AamTX	Z*VQTNKKCKGGSCASVCAKEIGVAAGKCINGRCVCYP	24
Aam-KTX	GVEINVKCTGSHQCIKPCKDAGMRFGKCINRKCHCTPK	21
AamBTX-L1	ADVPGNYPLDSSDNTYLCAPLGDNPDCIKICQKHGVDYGYCYAFQCWCEFLKDENVKV	22
AamBTX-L2	ADVPGNYPVDSSGKRYFCEPLGESKFCVDLCRKHGVKYGYCYLFMCWCEFLEDKNVKI	22
AamBTX-L3	ADVPGNFPLKPSRYRYSCPVLGDSEYVHVCRKHGVQYGYCWLFMCWCEYLEDKDVKI	22
AamAP1	FLFSLIPHAIGGLISAFK^#	23
AamAP2	FPFSLIPHAIGGLISAIK^#	23
AamAP-S1^$	FLFSLIPKAIGGLISAFK^#	23
*Z is pyroglutamyl residue; ^# Amidation; ^$Synthetic derivative of AamAP1 and AamP2.

Androctonus amoreuxi transcriptome:

We have assembled an A. amoreuxi venom gland transcriptome using Illumina shotgun whole transcriptome sequencing. The assembly is 95.4 Mb with a E90N50 of 2309 bp, > 96% of the evolutionary conserved arthropod BUSCO marker genes identified, and a high proportion of input reads map back to the assembly (> 98% all reads; > 96% strand-specific concordant pairs), indicative of a high-quality assembly (Supplementary Table 1).

Excluding 6 residual rRNA transcripts, a total of 102330 transcripts encoded by 75173 coding and noncoding genes were identified in the A. amoreuxi venom gland transcriptome (Table 2). Many transcripts in the A. amoreuxi transcriptome were expressed at very low levels (TPM < 1), reflecting the broad dynamic range of transcriptomes. These rare transcripts are expected to include many lncRNA but may also include some artefacts due to sequence error or misassembly (Supplementary Table 2). A total of 29062 transcripts from 25513 genes were expressed at higher levels (TPM > 1; mean TPM 30; median TPM 2), with 3819, 536 and 106 transcripts expressed at moderate (> 10 & <100 TPM), high (> 100 & <1000 TPM) or very high (> 1000 TPM) levels, respectively (Supplementary Table 2). Predicted peptides and proteins range in size from 99 to 5196 amino acids, with a mean and median length of 379 and 248 amino acids, respectively.

Table 2

Trinotate annotation metrics
Features	Total
Transcripts¹	102330
Genes²	75173
Non-coding²	59344
Protein coding²	15829
Novel proteins²	4205
Annotated transcripts³	10509
Arthropod³	5695
Scorpion³	513
Pfam Domains⁴	11922
Transmembrane Domains⁴	6443
Signal peptides⁴	2234
A. amoreuxi venom gland transcriptome was annotated with Trinotate and annotation metrics are shown.
1. Total number of predicted transcripts. This includes transcript variants and excludes rRNA. Libraries were enriched for polyadenylated transcripts and background rRNA transcripts were excluded post-assembly (n = 6).
2. Number of unique genes, excluding rRNA genes (n = 2) and including those predicted to be protein coding or putative non-coding RNAs not containing a predicted ORF. Protein coding genes include genes encoding novel proteins with no homologous protein in the NCBI protein database (no blast hits).
3. Number of transcripts with a homologue in the NCBI nucleotide and/ or protein database (blast), and the subset with highest identity to an arthropod or scorpion gene. Top blast hit is Arthropoda or Scorpion, respectively. Includes protein coding and non-coding genes.
4. Number of genes with functional or structural domains, or signal peptides, identified using HMMER/ Pfam, TmHMM or SignalP, respectively.

A total of 10509 A. amoreuxi transcripts were assigned biological functions from sequence identity with database homologues, and over half of these genes have highest identity with an arthropod gene (Table 2; Supplementary Table 3). Predicted proteins include 56 transposable element related proteins. Due to the limited genomic resources available for scorpions, along with poor annotation of many lncRNAs, identity assignment via homology with entities in the NCBI databases was not possible for all transcripts. However, 36671 transcripts had gene ontologies assigned from nucleotide sequence, providing broader functional annotation, and the protein products of over 2000 A. amoreuxi venom gland transcripts are predicted to have ion channel, transporter or toxin activity (Supplementary Table 3). Many putative novel proteins were identified in the A. amoreuxi transcriptome with 4205 unannotated genes containing an open reading frame (Table 2; Supplementary Table 3). Structural and functional domains, and signal peptide sequences were identified in transcripts with or without functional annotation (Table 2; Supplementary Table 3).

The dbAMP version 2.0 database contains 28709 predicted (n = 10364) or experimentally validated (n = 18345) antimicrobial peptides or proteins across a range of taxa, of which 1260 are arthropod derived, and 1803 are anti-viral peptides. The latter include 187 peptides with experimentally validated anti-SARS-CoV-2 or anti-coronavirus activity.²⁵ Screening of the A. amoreuxi transcriptome against the dbAMP 2.0 database identified many transcripts encoding putative AMP. This includes 16 A. amoreuxi genes encoding peptides, or containing peptide regions, with 100% identity to previously reported AMP (Supplementary Table 4). Many more A. amoreuxi transcripts are predicted to encode putative AMP through nucleotide sequence identity with known AMP (AMPFinder: 121 strict with > 90% identity and e-value < 1e-20; and 164 loose > 80% & < 90% identity alignments) (Supplementary Table 5). In addition, AMP-prediction models using the dbAMP 2.0 insect RF classifier, identified 3231 A. amoreuxi transcripts as encoding putative novel antimicrobial peptides (AMPFinder, Predictive score ≥ 90%, including 199 transcripts with a predictive score of 100%; Supplementary Table 6).

Chemistry

Table 3 shows a list of 15 new peptides that were isolated from the venom of A. amoreuxi with chain length spanning 12–65 amino acid residues. These peptides were identified based on their MS fragmentation pattern and the obtained transcriptomic data of A. amoreuxi. The sequences of the corresponding precursor peptides and the encoding transcripts with their abundances are also included (Table 3). The identified peptides can be categorized into three groups; the non-disulphide bridged peptides (AM29A1 and AM29A2), the short chain peptides with disulphide bridges (AM28, AM29A3, AM29A4 and AM29A5) and the long chain peptides with disulphide bridges (AM42, AM42A5-1, AM42A5-2, AM44A8-1, AM44AB-2, AM44A9, AM51A4, AM53A6 and AM53A7). The MS fragmentation pattern of these peptides is shown in the Supplementary Figs. 1–27 and the list of MS data is provided in the Supplementary Table 7. The MS analysis revealed a C-terminal amidation in AM44A8-2. A slight deamidation, which is most likely non-enzymatic, was also noticed in samples AM29A2, AM42A5-1, AM42A5-2 and AM51A4. In most cases, the signal peptide was deduced from the alignment of the precursor peptides with homologous peptides (Supplementary Figs. 3, 5, 7, 9, 11, 12, 15, 17–19, 21, 23, 26) and using SignalP.²⁶ CD-UV spectra were recorded for peptides AM42A5-1, AM44A9, AM53A6 (Supplementary Figs. 12, 19, 23). The limited quantities available for other peptides did not allow the acquisition of good spectra. A C-terminus amidated derivative of peptide AM29A5 was synthesized and correctly folded in buffer condition 4 (Table 4, Supplementary Fig. 28). The CD-UV data of the synthetic peptide AM29A5-syn was acquired and shown in Supplementary Fig. 30.

Table 4

Folding buffers used. Reduced glutathione (GSH). Oxidized glutathione (GSSG)
Buffer	Peptide aq. solution (2 mg/mL)	0.1 M NH₄HCO₃	Isopropanol	20 mM Reduced glutathione (GSH)	4 mM Oxidized glutathione (GSSG)
1	20 µL	130 µL	50 µL
2	20 µL	90 µL	50 µL	20 µL	20 µL
3	20 µL	40 µL	100 µL	20 µL	20 µL
4	20 µL	180 µL

Biological screens

Venom peptides as inhibitors of RBD binding to hACE2:

Surface plasmon resonance competition assays were used to evaluate the venom-derived peptides as inhibitors of RBD binding to ACE2. Eight peptides were selected for testing based on the findings of our initial screening of binding of partially purified samples to RBD (data not shown), namely AM28, AM29A3, AM29A4, AM29A5 (and the respective synthetic peptide AM29A5-syn), AM42A5-2, AM44A9, AM53A6 and AM53A7. Inhibition assays were carried out by immobilization of RBD on the sensor chip surface and binding measurement of fluid phase ACE2 at a fixed concentration in presence of pure peptides at varying concentrations.

Seven out of eight peptides showed some level of dose-dependent inhibition of ACE2 binding to RBD: AM29A3, AM29A4, AM29A5/ AM29A5-syn, AM42A5-2, AM44A9, AM53A6 and AM53A7. The inhibitory activity of two representative peptides (AM53A6 & AM29A5) is shown in Fig. 1. The IC₅₀ values ranged from 117 µM for AM29A5/ AM29A5-syn to 1202 µM for AM44A9, although the IC₅₀ could not be determined for AM29A3 and AM53A7 due to insufficient data points (Table 5). AM28 did not show dose-dependent inhibition in this assay at concentration as high 288 µM (data not shown). To measure the direct binding activity of the peptides to RBD, fluid phase peptides alone were injected at varying concentrations on immobilized RBD. Peptides AM29A5/ AM29A5-syn and AM53A6 showed dose-dependent binding to RBD with affinity of 280 µM and 3.3 mM, respectively (Table 5) as determined by sensorgram fitting to the 1:1 binding model. Peptides AM29A3, AM29A4, AM42A5-2, AM44A9 and AM53A7 demonstrated binding to RBD only at the higher end of the range of concentrations tested (Table 5). In these instances, binding kinetics could not be evaluated due to limitations on sample quantity. The binding activity of two representative peptides is shown in Fig. 1. Peptide AM28 did not bind RBD in keeping with the lack of activity of this peptide in inhibition assays (data not shown).

Table 5

RBD - hACE2 binding inhibition and RBD binding measurements of venom-derived pure peptides.
Name of peptide	Concentration range (µM)	RBD - hACE2 binding inhibition	Binding to RBD
AM28	28.8–288.0	no	no
AM29A3	43.4–434.0	IC₅₀ not determined (14% at 43.4 µM, 27% at 434 µM)	yes (detected only at 434 µM)
AM29A4	13.2–264.0	IC₅₀ = 175 µM	yes (detected at 264 µM)
AM29A5/AM29A5-syn	2.5–245.0	IC₅₀ = 117 µM	Dose-dependent binding k_a (M^− 1 s^− 1) = 14.8 k_d (s^− 1) = 4.1E^− 3 K_D (M) = 2.8×10^− 4 Chi2 = 8.45
AM42A5-2	36.7–550.5	IC₅₀ = 838 µM	yes (at ≥ 367 µM)
AM44A9	17–340	IC₅₀ = 1202 µM	yes (detected at 340 µM)
AM53A6	6.5–130	IC₅₀ = 328 µM	Dose-dependent binding k_a (M^− 1 s^− 1) = 4.8 k_d (s^− 1) = 1.5×10^− 3 K_D (M) = 3.3×10^− 3 Chi2 = 12.2
AM53A7	6.3–63	IC₅₀ not determined (16% at 6.3 µM, 30% at 63 µM)	yes (detected at 63 µM)

Antiviral activity of peptide AM29A5syn:

We undertook SARS-CoV-2 neutralisation assays in Vero E6 cells to assess the inhibitory activity of peptide AM29A5syn against replication-competent virus. On virus exposure to AM29A5-syn, we observed a two log₁₀ PFU/mL reduction at sub-micromolar concentrations of peptide compared to virus exposed to medium alone (Fig. 2). TCID50/mL in the presence and absence of peptide was 7.03×10² and 1.25×10⁴, respectively. In MTT dye reduction assays, there was no evidence of reduced Vero cell viability on exposure to the AM29A5-syn peptide at concentrations as high as 1.6 mM (data not shown).

In this manuscript, we identified 15 new peptides from the venom of the scorpion, A. amoreuxi (Table 3) using MS fragmentation and venomous gland transcriptome mining. To the best of our knowledge, only 10 peptides have been previously identified from the venom of A. amoreuxi (Table 1) and the transcriptome or genome of this species has not yet been published. We tested the ability of these peptides to inhibit the critical interaction between the SARS-CoV-2 spike RBD and hACE2 that precedes virus entry into the cell⁵ using a surface plasmon resonance-based assay. Several scorpion venom peptides are conformationally constrained with a set of disulfide bridges between Cys residues.²⁷ These disulfide bridges allow the peptide to fold into the correct 3D conformation that can interact with the receptor. Most disulfide bridge-containing peptides interact specifically with neuronal ion channels, and they are further classified into four families based on the ion channels they target (Na⁺, K⁺, Cl⁻ or Ca²⁺ channels). Sodium channel-specific toxins are usually long-chain peptides with 60–76 amino acid residues in length and are cross-linked with three or four disulfide bridges.^{27, 28} Potassium channel-specific toxins are made up of peptides with 23–64 residues with three or four disulfide bridges²⁹ while chloride ion channel-specific toxins are composed of fewer than 40 amino acid residues and contain four disulfide bonds.³⁰ However, recent research has demonstrated that constrained peptides e.g. macrocyclic or stapled peptides can inhibit disease relevant protein-protein interactions such as SARS-CoV-2 Spike-hACE2, which are known to be very challenging to small molecule drugs.^{31, 32} Moreover, the antiviral activity of several scorpion venom peptides against a wide range of viruses including SARS-coronavirus have been confirmed and several mechanisms have been suggested.^9–17 A recent molecular docking study³³ has revealed the ability of some known antiviral scorpion venom peptides to interact with the SARS-CoV-2 spike RBD. However, no supporting experimental biological data were provided. These studies and the lack of knowledge on the venom peptides from A. amoreuxi were the stimulus for this research.

We report the first annotated reference transcriptome for the A. amoreuxi venom gland. Transcriptome annotation and AMP mining have identified a wealth of diverse A. amoreuxi venom gland peptides with putative ion channel, transporter or toxin activity, as well as putative orthologues of known AMPs and predicted novel AMPs. This catalogue of potentially medically important gene products provides rich data to support antimicrobial drug development. Furthermore, our A. amoreuxi transcriptome has enabled a proteotranscriptomic approach to antiviral drug discovery, through identification of full-length transcripts encoding SARS-CoV-2 spike protein inhibitors, that were partially resolved by MS fragment sequences.

Out of the 15 venom peptides identified in this study, eight were tested for their ability to bind to SARS-CoV-2 spike protein and to inhibit its interaction with hACE2 (Table 5, Fig. 1). Peptides AM29A4 and AM29A5 displayed the highest inhibition and lowest IC₅₀ whilst AM28 showed no evidence of inhibition. Peptide AM53A7 also showed notable inhibition at relatively low concentrations whilst the effect of this peptide at higher concentrations could not be determined due to limited amount of sample.

This data aligned with findings of direct binding to RBD, notwithstanding that full binding kinetics data could only be determined for peptides AM29A5 and AM53A6. The properties of most highly active peptide AM29A5 were confirmed by testing the chemically synthesized analogous peptide which displayed the same IC₅₀ and RBD-binding affinity values as AM29A5. Altogether these findings provide evidence of specific and competitive inhibition of RBD - hACE2 binding by seven of the peptides tested likely occurring, at least in part, through interaction with the hACE2 binding site of the SARS-CoV-2 spike RBD. SARS- CoV-2 RBD - hACE2 is a very challenging target for drug design e.g. in a previous study,³⁴ a series of 13 different stapled peptidomimetics based on the ACE2 interaction motif were designed to bind the coronavirus S-protein RBD and inhibit binding to the hACE2 receptor. The peptidomimetics were assessed for antiviral activity in an array of assays including a neutralization pseudovirus assay, immunofluorescence (IF) assay and in vitro fluorescence polarization (FP) assay. However, none of the peptidomimetics showed activity in these assays. Importantly, this highlights the importance of developing an effective strategy to screen biological resources, like scorpion venoms, with proven therapeutic potential in combating microbial and viral pathogens, to identify and test putative antiviral peptides, thus providing evidence-based lead candidates for further exploration and development. The antiviral activity of AM29A5-syn was seen at a concentration below the concentration range used in RBD - hACE2 surface plasmon resonance assay, suggesting an additional antiviral mode of action by this peptide.

Collectively, our results show that scorpion venom peptides may have the ability to inhibit SARS-CoV-2 spike protein interaction with hACE2 receptor which precedes the virus entry to the cells while exhibiting anti-SARS-CoV-2 activity via additional unexplored modes of action. These peptides could be used as a blueprint to design natural product analogues that are more potent, highly selective and less toxic inhibitors to this COVID-related key protein-protein interaction.

Compliance with the Nagoya protocol on Access and Benefit Sharing

This research project was conducted in accordance with the obligations under the Nagoya Protocol on Access and Benefit Sharing. We obtained Prior Informed Consent (PIC) no. 00308021010800/4 from the Egyptian Convention on Biological Diversity (CBD) National Focal Point to conduct this research using the scorpion, A. amoreuxi collected from Egypt.

Collection of Scorpions and Venom Preparation

Adult A. amoreuxi scorpions were purchased from El Marwa Office for Export (Egypt) and were housed individually (to avoid cannibalism) in clear plastic containers in the laboratory of Professor Abdel-Rahman at Suez Canal University. The scorpion specimens were identified according to the key of El-Hennawy.³⁵ Scorpions were fed on small insects (mealworms) and received water ad libitum. Crude venom was extracted using electrical stimulation (12–16 V; 10 s) and the milked venom was collected and centrifuged for 20 min at 13,000 rpm/4°C. Clear supernatants were pooled, freeze-dried and stored at − 20°C until use.³⁶

Illumina sequencing

RNA was extracted from telsons dissected from five adult scorpions and stored in RNAlater® at − 80°C. Telsons were shipped on dry-ice and stored on receipt at − 80°C until processing. Scorpions were anesthetized before telson dissection by putting them in a freezer at − 20°C for ∼4–5 minutes. Venom was milked 4 days before dissection as recommended in previous studies.^36–38 Telsons were combined, washed in ice-cold PBS to remove RNAlater® and homogenized in 1 mL TRIzol reagent (Thermo Scientific, UK) with 2 x 3mm tungsten carbide beads in a TissueLyser II (Qiagen, CA). Total RNA was extracted in TRIzol (Thermo Scientific, UK) and purified on RNeasy micro columns with on column DNase digestion (Qiagen, CA), according to the manufacturer’s instructions. Total RNA was quantified by fluorimetry (Qubit, Thermo Scientific, UK) and quality (RIN 9.1) was assessed on a Tapestation 4200 (Agilent, CA). Unique dual indexed stranded TruSeq mRNAseq libraries were prepared from 500 ng total RNA, according to the manufacturer’s instructions (Illumina, CA). Libraries were quantified by qPCR with SYBR green (Kapa Library Quantification Complete Universal, Roche, CH) on the QuantStudio 6 Flex (ThermoFisher Scientific, UK) and average library size of 291 bp determined on the Tapestation 4200 (Agilent, CA) prior to sequencing and base calling on an Illumina NextSeq500 with v2.5 chemistry and 75 bp paired-end reads, with 14.3 Gb sequence output.

Transcriptome Assembly and Annotation

Raw read quality was assessed before and after pre-processing with FastQC (version 0.11.8)³⁹ and MultiQC (version 1.7)⁴⁰. Raw reads were pre-processed using Trim Galore! (Version 0.6.4)⁴¹ to trim low-quality bases (< Q30) and adapters, and excluding short trimmed reads (< 20bp), before de novo transcriptome assembly with Trinity (version 2.8.5),^{42, 43} with unique accession Trinity_DN prefix identifying each feature in the output assembly. Assembly quality was assessed with BUSCO (version 4.0.6) with the arthropod lineage dataset, arthropoda_odb10,⁴⁴ rnaQUAST (version 2.2.0)⁴⁵ and GeneMarkS-T (version 5.1) gene prediction.⁴⁶ Transcript abundances were determined with embedded Trinity scripts using the salmon method.⁴⁷ For evaluation of Trinity transcripts against MS identified peptides, input raw reads used for the venom gland transcriptome assembly were mapped against the assembly using Bowtie 2 (version 2.3.5).⁴⁸ The alignment file was post-processed with SAMtools (version 1.9) and coverage and allelic composition of transcripts of interest were inspected with SAMtools mpileup (version 1.9),^{49, 50} with corresponding sequence logos produced with R (version 3.6.1),⁵¹ Rstudio (version 1.1.456),⁵² ggplot2 (version 3.3.6),⁵³ and ggseqlogo (version 0.1)⁵⁴.

The assembly was annotated using Trinotate software (version 3.2.1).⁵⁵ Residual background rRNA transcripts were filtered in post-assembly QC using Trinotate RNNAMER classification. AMPFinder with Diamond (version 1.1.0) was used to identify putative A. amoreuxi antimicrobial peptide orthologues of known AMPs with the A. amoreuxi contig nucleotide sequences as input queries against translated RNA sequences of known AMPs in the manually curated dbAMP version 2.0 database.²⁵ Additionally, AMPFinder (version 1.1.0) random forest (RF) classifiers were used to discover putative novel AMPs in the A. amoreuxi transcriptome²⁵. Jhong et al.²⁵ constructed dbAMP 2.0 RF classifiers using experimentally verified AMPs from different source organisms (bacteria, human, amphibian, fish, plants, insects and mammals) as distinct training and test datasets to develop RF models with the best AMP-predictive performance in each lineage, as well as accurate predictive performance of each across all lineages (> 92% for independent test data). Prediction scores in insects, representing the most closely related taxa to scorpions, are reported for A. amoreuxi.

Isolation of venom peptides

Reverse phase high performance liquid chromatography (RP-HPLC) was performed using an Agilent 1260 LC System. Forty milligrams of the crude venom were dissolved in 4 mL (50:50 water: acetonitrile v/v), cleared by centrifugation at 7,000g and the supernatant was injected into a C4 column (ACE 5 C4-300, 5 µm, 250 × 10 mm, 300 Å). The fractions were then further purified using an C18 column (ACE 5 C18-300, 5 µm, 250 × 10 mm, 300 Å). Peptides were eluted using a linear gradient starting at 60% of solution B (0.1% TFA in acetonitrile) in solution A (0.1% TFA in water) for 60 min with a flow rate of 3 mL/min. Individual fractions were manually collected based on the peak absorbances at 220 nm, 254 nm and 280 nm. The collected samples where then dried using rotary evaporator to remove the acetonitrile, followed by lyophilization using a LaboGene CoolSafe freeze dryer.

Mass spectrometric analysis

MS data was acquired on a 12T SolariX 2XR Fourier transform – Ion cyclotron resonance (FT-ICR) instrument equipped with electrospray (ESI) ionization (Bruker Daltonics). RP-HPLC fractionated samples were infused at 2 µL/min and spectra were acquired between 200 and 4000 m/z. For top-down fragmentation, individual ions (single proteoforms) were isolated using the quadrupole and tandem MS was performed using either collision induced dissociation (CID) or electron capture dissociation (ECD). For ECD, typical cathode conditions were a bias of 1.5 V, lens voltage of 15 V, and pulse length was varied between 5 and 15 ms. Collision induced dissociation (CID) was typically conducted using a voltage between 15–25 V. The resulting top-down fragmentation spectra were processed using Data Analysis v4.2 (Bruker Daltonics) and the sophisticated numerical annotation procedure (SNAP) was used to produce a monoisotopic mass list. These mass lists were manually searched for sequence tags. Translated transcripts were searched for these sequence tags to identify the encoding transcript of each peptide and the sequence of the precursor peptide using the sequence logo method described above.

Alignment and phylogenetic tree

Protein homologues of each MS-derived peptide sequence were identified in the NCBI protein database and aligned using NCBI-Blast. Phylogenetic trees were produced using the bootstrap method with 100 bootstrap replications with MEGA11.⁵⁶

Template-based 3D modelling

The homology modelling of the identified peptides with more than 30 amino acid residues was performed using SWISS-MODEL.⁵⁷ The templates were selected based on the global model quality estimate (GMQE) which is a quality estimate that combines properties from the target-template alignment and the template structure. For large peptides (> 50 aa), we used GMQE cut-off value of 0.6, coverage = 0.9 and identity = 40% while the corresponding values for short peptides (< 50 aa) are GMQE = 0.8, coverage = 0.9 and identity = 80%. Visualisation and drawing of selected models were executed with PyMOL. The presence of disulfide (DS) bridges was confirmed through MS analysis, and the configuration of these DS bridges was predicted through homology with previously identified scorpion toxins.

Far-UV circular dichroism (CD): CD spectra of purified peptides (50 µM in 20 mM sodium phosphate buffer [pH 7.4] and 20 mM sodium fluoride at 25°C) were acquired between wavelengths of 260 nm and 190 nm on a MOS-500 CD Spectrometer (BioLogic). Each CD spectrum represent the average of 3 scans which was baseline-corrected by subtracting of the buffer spectrum. Mean residue differential extinction coefficient Δεres of each spectrum was calculated from the observed ellipticity θ and plotted against wavelength. The CD analysis was carried out only on peptides with sufficient amounts. Bestsel server (https://bestsel.elte.hu/index.php) was employed for determining the secondary elements from CD spectra.⁵⁸

Solid phase peptide synthesis (SPPS):

Reagents

The majority of the Fmoc amino acid derivatives were purchased from Novabiochem, Merck Biosciences, UK. Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, N,N’-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were acquired from Fluorochem, UK. Fmoc-L-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, ethyl cyano(hydroxyimino)acetate (Oxyma), Fmoc-Rink Amide ProTide (LL) resin was obtained from the CEM corporation, USA. N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (MeOH) and diethyl ether were purchased from VWR, Avantor, USA. Piperidine was purchased from Merck Life Sciences, UK. Sodium diethyldithiocarbamate trihydrate, 2,2′-(ethylenedioxy)diethanethiol (DODT) and pyridine were acquired from Sigma Aldrich.

Synthesis: Peptide synthesis was performed on a Liberty BlueTM Automated Microwave Peptide Synthesizer (CEM Corporation, North Carolina, USA) using the standard solid-phase peptide synthesis (SPPS)-Fmoc/tBu chemistry with piperidine (20% v/v) as the Fmoc-deprotecting agent. The AM29A5-syn peptide was assembled on a 0.1 mmol scale using Fmoc-Rink Amide ProTide (LL) resin with a substitution value of 0.18 mmol/g using 0.2 M Fmoc-protected amino acids and activated by DIC (1 M) and in the presence of 1 M Oxyma prepared in DMF. All the arginine residues were double coupled, and rest of the amino acids were single coupled. On synthesis completion, the resin was washed with dry DCM to remove DMF and subsequently dried under N₂. The dried-resin peptide was cleaved with a cocktail solution of TFA/TIS/DODT/H₂O (92.5:2.5:2.5:2.5) for 4 hours followed by filtration to remove resin beads. The flow through was then concentrated under N₂ gas. The peptides were precipitated using cold diethyl ether, placed into a − 20ºC freezer overnight, washed with ether (3x) and dried under vacuum to give a crude solid. The crude peptides were purified using reversed phase HPLC on an Agilent Technologies 1260 Infinity using a C18 column (ACE 5 C18-HL, 5 µm, 250 × 10 mm, 100 Å) through an acetonitrile (+ 0.1% TFA)/Water (+ 0.1% TFA) gradient. Fractions containing the pure peptide were subsequently lyophilized on a LaboGene CoolSafe Freeze dryer to give the pure solid. Peptide identity was confirmed by MS analysis.

Folding

Four buffers (Table 4) were used to allow the correct oxidative folding of the linear synthetic peptide (AM29A5-syn). Aliquots of each reaction were collected at different time intervals and immediately quenched using 0.4% TFA in MilliQ water. The quenched aliquots were then injected in a C18 column (ACE 5 C18-300, 5 µm, 250 × 10 mm, 300 Å) and the retention time of the folded product was compared with that of the native peptide. MS analysis was also used to confirm the formation of DS bridges.

Surface plasmon resonance:

Recombinant proteins

The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 (aa 319–541) and human ACE2 (aa 19–615) were recombinantly produced in HEK293 cells and provided by Peak Proteins Ltd (Macclesfield, UK). QC sheets are provided (Supplementary document 8 & Supplementary document 9).

Surface plasmon resonance assays

Venom peptide binding to RBD and competition with hACE2 was assayed in surface plasmon resonance experiments carried out using Biacore X100 (Cytiva, Uppsala, Sweden).

RBD binding measurements: RBD in PBS-P + pH 7.4 was captured and immobilized (approximately 6300 RU) on the surface of Flow cell 2 of an NTA sensor chip using the standard nickel activation procedure (Cytiva). This was followed by activation with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 7 min and inactivation with ethanolamine for 7 min for covalent linking of the 6×His-tag-RBD to the nickel-activated surface. Flow cell 1 (activated and blocked) served as a reference (blank) cell. Binding of fluid phase venom-derived peptides was determined over a range of concentrations determined by peptide quantity. Peptides were dissolved in PBS-P + buffer pH 7.4 (0.2 M phosphate buffer, 27 mM KCl, 1.37 M NaCl, 0.5% surfactant P20; Cytiva), supplemented with 71 µL of 350 mM EDTA. Injection volume was 60 µL and flow rate was 5 µL/min. The surface was regenerated with 350 mM EDTA and 0.5% w/v SDS. Sensorgrams of RBD binding measurements were obtained by subtraction of the sensorgram of buffer alone from that generated by injection of peptides. Equilibrium dissociation constants (K_D) as well as association (k_a) and dissociation constant (k_d) rates were calculated for the peptides that showed dose-dependent binding using the Biacore X100 Evaluation Software version 2.0.2 (Cytiva). Curves were fitted to a 1:1 binding model as judged by the Chi² value and distribution of residuals.

Inhibition studies

Binding of fluid phase hACE2 at a concentration of 150 nM was determined in the presence of venom peptides at the same concentrations listed above and compared to binding activity of hACE2 alone. For peptides that showed direct binding activity to RBD, final inhibition sensorgrams were obtained by subtraction of the sensorgram of injection of peptides alone from that generated by injection of peptides in presence of hACE2. Fluid phase compounds were dissolved in PBS-P + pH 7.4 buffer supplemented with 500 µM EDTA. Flow rate was 5 µL/min and the injection volume was 60 µL. After each binding measurement, the surface was regenerated with 250 mM EDTA and 0.5% w/v SDS. Single measurements were carried out for each condition. The half-maximal inhibitory concentration (IC₅₀) was defined as the concentration of peptide that reduced hACE2 binding to RBD to 50% when compared with binding of hACE2 alone. IC₅₀ values were calculated by linear regression using Microsoft Excel.

Antiviral assay:

Cells, virus and reagents

Vero E6 cells (ATCC-CRL-1586) for SARS-CoV-2 propagation, antiviral assays and cytotoxicity assays were obtained from LCG Standards (Middlesex, UK). Cells were grown and maintained in grown in Dulbecco's Modified Eagles Medium (DMEM) (Sigma, UK) supplemented with 10% foetal calf serum, 1 mM Sodium Pyruvate (Gibco) and 2 mM L-glutamine (Sigma). Cells were grown in an environment enriched with CO₂ (5%) at 37°C and passaged every 2–3 days or at approximately 80% confluence; the number of passages did not exceed 6.

SARS-CoV-2, isolate hCov-19/England/204820464/2020 (Lineage B.1.1.7) was kindly made available by Public Health England through the BEI resources repository National Institute of Allergy and Infectious Diseases and managed by ATCC. The virus was propagated in Vero cells for 72 h at 37°C and 5% CO₂ in DMEM supplemented with 2% FCS before recovery and storage (− 70°C) of cell-free virus. Viruses from second and third cell passages were used in all experiments. Infectivity was estimated by measurement of 50% tissue culture infective dose (TCID50) values of the cell-free viral supernatants.

The anti-RBD human neutralising antibody CV30,⁵⁹ used as reference compound in antiviral assays, was purchased from Absolute Antibody (Cleveland, UK).

Cytotoxicity assays

Peptide cytotoxicity was assessed using the Vybrant® MTT Cell Proliferation Assay Kit (Thermofisher Scientific) by comparison of cell viability in the presence vs absence of peptide. Vero cells were seeded at 3 × 10⁴ per well for 24 h. Cells were then washed with PBS and challenged with 2-fold dilutions of the AM29A5-syn peptide diluted in DMEM for 24 h at the range of concentrations tested in antiviral assays. After 24 h, the supernatant was removed and cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in 100 µL of DMEM (free of phenol red) for 4 h at 37°C in 5% CO₂. Finally, 100% DMSO was added for formazan dissolution at 37°C before absorbance assessment at 540 nm. Viability was estimated by comparison with cells exposed to medium alone and assays were performed in triplicate.

Virus neutralisation assays

Vero cells were seeded at 3 × 10⁴ per well in DMEM supplemented with 10% FCS and incubated for 24 h at 37°C and 5% CO₂. On the day of the experiment Vero cells were challenged with 10-fold serial dilutions of cell-free SARS-CoV-2 (starting concentration 10⁸ PFU/mL) pre-incubated with 2-fold serial dilutions of AM29A5-syn peptide (0.8 µM – 25 µM) in DMEM with 2% FCS for 1h. The neutralising antibody CV30 at 2 µg/mL and medium alone were used as positive and negative control, respectively. After the 1 h pre-incubation period, 2.5% Avicell (Sigma) in 100 µl of water was added before the main incubation for 72 h at 37°C and 5% CO₂ to allow for viral replication. After this time, infected cells were fixed with 10% formalin (Sigma) for 3 h, before staining with crystal violet in 20% ethanol (Sigma) for 30 min. Finally, wells were washed two times with water and left to dry before plaque counting under light microscopy for quantification of plaque-forming units (PFU). Infectivity was estimated by TCID50 measurements and comparison with cells exposed to medium alone. Each condition was performed in quadruplicate.

ACKNOWLEDGEMENTS

This project was supported by a fellowship grant from the EPSRC (No. EP/S027246/1, W. E. H.), and a grant from the Global Challenges Research Fund (GCRF) Covid-19 Response Scheme (W. E. H., E. C. D., K. H., M. A. A.). We are very grateful to Prof Moustafa M. Fouda, the CBD National Focal point of Egypt for providing the PIC to conduct this research using the scorpion, A. amoreuxi collected from Egypt. We would like to thank Prof Samy Zalat for his valuable advice on the application process to the CBD National Focal point of Egypt. We would like to thank Dr Farhan Ali and Dr Magnus Alphey, University of St Andrews for their help with the CD analysis of our peptides. We would like to acknowledge the support of Digital Research and the Maxwell high performance compute cluster, University of Aberdeen.

AUTHOR CONTRIBUTIONS

W. E. H., M. A. A., K. H. and E. C. D. designed the research. M. A. A., R. S. and M. M. G. collected the scorpion venom. A. G., D. C. and W. E. H. purified and identified the venom peptides. A. R., Z. H., and E. C. D. carried out the transcriptomic experimental work and analysis. C. P., K. B. and K. H. developed the surface plasmon resonance-based assay. T. M., A. G. and S. A. synthesized the venom peptide. R. A. C., K. S. S. and K. H. carried out the cytotoxicity and antiviral assay. W. E. H., A. G., D. C., K. H., T. M. and E. C. D. wrote the first draft of the paper.

Data availability statement

The A. amoreuxi transcriptome data is available at GEO. Accession number: GSE221799.

Competing interests

The authors declare no competing interests.

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Table 3 is available in the Supplementary Files section.

No competing interests reported.

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Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS- CoV-2 activity

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Abstract

Figures

Introduction

Results

Chemistry

Biological screens

Venom peptides as inhibitors of RBD binding to hACE2:

Antiviral activity of peptide AM29A5syn:

Discussion

Methods

Solid phase peptide synthesis (SPPS):

Surface plasmon resonance:

Antiviral assay:

Declarations

References

Table

Additional Declarations

Supplementary Files

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