RESEARCH Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling Authors Cara F. Smith, Cassandra M. Modahl, David Ceja Galindo, Keira Y. Larson, Sean P. Maroney, Lilyrose Bahrabadi, Nicklaus P. Brandehoff, Blair W. Perry, Maxwell C. McCabe, Daniel Petras, Bruno Lomonte, Juan J. Calvete, Todd A. Castoe, Stephen P. Mackessy, Kirk C. Hansen, and Anthony J. Saviola Correspondence Graphical Abstract 2024, Mol Cell Proteomics 23(6), 100 © 2024 THE AUTHORS. Published b Molecular Biology. This is an open a creativecommons.org/licenses/by-nc https://doi.org/10.1016/j.mcpro.2024 Anthony.Saviola@cuanschutz. edu In Brief Smith et al. investigate the utility of the proteome integral solubility alteration (PISA) assay for elucidating venom protein interactions with a small molecule inhibitor. Thermal proteome profiling experiments demonstrate that much of the Western Diamondback rattlesnake (Crotalus atrox) venom proteome is amenable to thermal denaturation. PISA identified specific SVMP proteoforms targeted by the matrix metalloprotease inhibitor marimastat, demonstrating that a PISA-based approach can provide rapid, highly sensitive, and robust inferences for the unbiased proteome-wide screening of venom and inhibitor interactions. Highlights • A significant proportion of the venom proteome is amenable to thermal denaturation. • The PISA assay can identify venom protein targets of small molecule inhibitors. • Both the supernatant and pellet provide useful and complementary information. • Optimizing the thermal window based on the Tm of the target protein improves results. 779 y Elsevier Inc on behalf of American Society for Biochemistry and ccess article under the CC BY-NC-ND license (http:// -nd/4.0/). .100779 mailto:Anthony.Saviola@cuanschutz.edu mailto:Anthony.Saviola@cuanschutz.edu http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ https://doi.org/10.1016/j.mcpro.2024.100779 http://crossmark.crossref.org/dialog/?doi=10.1016/j.mcpro.2024.100779&domain=pdf RESEARCH Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling Cara F. Smith1, Cassandra M. Modahl2 , David Ceja Galindo1 , Keira Y. Larson1 , Sean P. Maroney1, Lilyrose Bahrabadi1 , Nicklaus P. Brandehoff3 , Blair W. Perry4 , Maxwell C. McCabe1, Daniel Petras5,6 , Bruno Lomonte7 , Juan J. Calvete8 , Todd A. Castoe9 , Stephen P. Mackessy10 , Kirk C. Hansen1, and Anthony J. Saviola1,* New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin in- hibitors have shown promising cross-species neutraliza- tion of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and out- comes of snakebite envenomation worldwide. Energetics- based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent “deep proteomics” methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and charac- terize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA ther- mal window and soluble supernatant with insoluble pre- cipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high- throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom. From the 1Department of Biochemistry and Molecular Genetics, Univers Research and Interventions, Liverpool School of Tropical Medicine, Liv and Hospital Authority, Denver, Colorado, USA; 4School of Biologica 5CMFI Cluster of Excellence, University of Tuebingen, Tuebingen, Ger Riverside, California, USA; 7Instituto Clodomiro Picado, Facultad de 8Evolutionary and Translational Venomics Laboratory, Consejo Superi of Biology, The University of Texas Arlington, Texas, USA; 10Departm Colorado, USA *For correspondence: Anthony J. Saviola, Anthony.Saviola@cuanschutz © 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Bio This is an open access article under the CC BY-NC-ND license (http://creativecommons Snakebite is a global public health problem that dispro- portionately affects impoverished communities in rural tropical and subtropical regions. Annual estimates suggest that snakebite affects 1.8 to 2.7 million people worldwide, causing >138,000 deaths and leaving an even larger number of victims suffering permanent disabilities (1), which has led to the designation of snakebite as a neglected tropical disease by the World Health Organization (2, 3). Snake venoms are complex toxic cocktails of proteins and peptides derived from more than a dozen gene families, many of which have un- dergone duplication to generate multiple functionally diverse paralogs and associated proteoforms in the venom of a single species (4–6). While substantial variation exists in the relative mass and functional activity of venom proteins and peptides, most of these toxins have evolved to target and disrupt numerous bodily systems (7–10). Adding to the complexity of snake venoms, the most medically relevant toxin families tend to be the most diverse with many paralogs and associated proteoforms displaying moderate-to-high sequence similarity but in many cases exhibiting a spectrum of distinct biological effects (7, 9, 11–13). One of these families, snake venom metalloproteases (SVMPs), is ubiquitous across snake spe- cies (but is particularly abundant in viperid venoms) and is responsible for many of the life-threatening pathologies that result from snake envenomation, including local and systemic hemorrhage and tissue destruction (14–18). ity of Colorado Denver, Aurora, Colorado, USA; 2Centre for Snakebite erpool, UK; 3Rocky Mountain Poison and Drug Center, Denver Health l Sciences, Washington State University, Pullman, Washington, USA; many; 6Department of Biochemistry, University of California Riverside, Microbiología, Universidad de Costa Rica, San José, Costa Rica; or de Investigaciones Científicas (CSIC), Valencia, Spain; 9Department ent of Biological Sciences, University of Northern Colorado, Greeley, .edu. Mol Cell Proteomics (2024) 23(6) 100779 1 chemistry and Molecular Biology. .org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.mcpro.2024.100779 Delta:1_surname Delta:1_given name https://orcid.org/0000-0003-2211-3930 Delta:1_surname https://orcid.org/0000-0001-6544-8648 Delta:1_given name https://orcid.org/0000-0002-7845-0381 Delta:1_surname Delta:1_given name https://orcid.org/0009-0000-9526-7223 Delta:1_surname https://orcid.org/0000-0002-6140-5963 Delta:1_given name https://orcid.org/0000-0003-4905-7852 Delta:1_surname Delta:1_given name https://orcid.org/0000-0002-6561-3022 Delta:1_surname https://orcid.org/0000-0003-2419-6469 Delta:1_given name https://orcid.org/0000-0001-5026-3122 Delta:1_surname https://orcid.org/0000-0002-5912-1574 Delta:1_given name https://orcid.org/0000-0003-4515-2545 Delta:1_surname Delta:1_given name https://orcid.org/0000-0001-6890-512X mailto:Anthony.Saviola@cuanschutz.edu http://crossmark.crossref.org/dialog/?doi=10.1016/j.mcpro.2024.100779&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ https://doi.org/10.1016/j.mcpro.2024.100779 Target Specificity of Marimastat Against Snake Venom Toxins The substantial morbidity and mortality resulting globally from snakebite may seem surprising considering that anti- venoms (whole IgG molecules, Fab or F(ab′)2 fragments from venom-immunized animals) are often highly effective at recognizing and neutralizing the major toxic components of a venom (19, 20). A major challenge to antivenom efficacy, however, is the significant variation in venom composition that occurs at phylogenetic (21–26), ontogenetic (27–30), and geographic or population scales (31–36). As a consequence, antivenoms are most effective against the snake species whose venom was utilized during production and are often inadequate at recognizing venom components of different or even closely related snake species (25). Further, geographic venom variation may even result in poor neutralization within the same species when snakes used in antivenom production were sourced from a different location (33). Antivenoms also tend to be more effective at neutralizing systemic effects but less effective at neutralizing anatomically localized manifes- tations of envenomation, which can result in permanent tissue damage and disfigurement (1, 37–41). The storage, accessi- bility, and administration of antivenom also pose significant practical challenges in rural areas where it is needed most (10, 37, 42–48). These hurdles are further compounded by the excessive effort and cost of producing antivenoms for any one geographically-relevant set of venomous snake species. While the use of polyvalent antivenoms has been the mainstay treatment for snake envenomation, the development of non-immunological treatments that circumvent the limita- tions of antivenoms has been prioritized as a goal to address the impacts of snake envenomation globally by the World Health Organization (3). Recent applications of small molecule inhibitors against medically significant toxins have yielded promising preclinical results and these inhibitors have broad potential as supplemental therapies in combination with standard treatments (37, 49–54). These inhibitors have a number of advantages over current antivenom therapies including better peripheral tissue distribution, higher shelf stability, a higher safety profile, the ability for pre-hospital oral or topical administration, and greater affordability (55). The use of novel high-throughput approaches for the testing of venom toxin inhibitors and the identification of their targets could accelerate the implementation of effective small molecule in- hibitors, with the long-term potential of improving the treat- ment and outcome of snakebite envenomation globally. Numerous studies have examined the effects of various small molecule inhibitors on the biological activities of venoms in vitro and the neutralization capacity of these inhibitors in vivo (49–51, 53, 56–59). A repurposed low-specificity matrix metalloprotease inhibitor, marimastat, has shown effective neutralization of SVMP-rich venoms across multiple venomous snake species by preventing both local and sys- temic toxicity (53), decreasing hemotoxic venom effects (50, 51, 53, 60, 61), reducing SVMP-induced cytotoxicity (56, 60), and inhibiting extracellular matrix degradation (62). Because 2 Mol Cell Proteomics (2024) 23(6) 100779 marimastat has previously progressed to clinical trials as a cancer treatment, its safety profile has already been deter- mined, accelerating its development as a potential snakebite treatment (63, 64). When administered with other small molecule inhibitors, marimastat has shown in vivo neutraliza- tion of lethal toxicity and dermonecrosis in murine models (50, 56). Each of these studies assesses the downstream effects of inhibitor action in vivo or in vitro by measuring changes to biological activity or survival; however, to our knowledge, no studies exist using a direct assessment of venom-wide target– ligand interactions between venom toxins and small molecule inhibitors. Thermal proteome profiling (TPP) is a prominent energetics- based proteomic approach for identifying the molecular tar- gets of drugs. TPP builds upon the concept that a protein’s physicochemical properties are altered through interactions with extrinsic factors (e.g., other proteins, therapeutic drugs, metabolites) making it more or less resistant to thermal- induced denaturation (65, 66). Traditional TPP assays were centered on the principle that unbound proteins tend to denature and become insoluble when subjected to increasing temperatures, whereas proteins stabilized through in- teractions with extrinsic factors often exhibit increased ther- mal stability and remain in solution (65, 67–69). Identifying and quantifying such solubility changes via mass spectrometry can be used to infer direct or indirect interactions between a given compound and its protein targets (70). Recently, the proteome integral solubility alteration (PISA) assay has emerged as a powerful strategy that retains the breadth and sensitivity of TPP but with a significant reduction in sample preparation and analysis time (70, 71). PISA repre- sents a “deep proteomics” method for high throughput proteome-wide target identification of ligands, with improved target discovery and higher statistical significance for target candidates (70–72). In general, PISA experiments measure changes in protein precipitation that can be induced by altering temperature (70), solvent concentration (67), me- chanical stress (73), or ion concentration (74). In a thermal PISA assay, samples are subjected to heat across a temper- ature gradient (as in TPP) but are subsequently pooled prior to analysis (70, 71). Rather than generating melt curves to determine exact melting temperatures, PISA compares overall abundance of each measured peptide between controls and treatments to detect differences in melting properties when a compound of interest is added. This methodology allows multiple variables to be altered simultaneously (e.g., concen- tration, temperature) in a high-throughput manner. It has recently been shown that heat-treating within a smaller tem- perature window can improve sensitivity and target discovery with PISA (75). TPP, PISA, and related methods derived from the same principles have been used to discover drug targets, antibiotic targets, mechanisms of antibiotic resistance, and in the high-throughput screen of compound libraries (65, 66, 69, 76, 77). In our specific context, these proteomic techniques Target Specificity of Marimastat Against Snake Venom Toxins applied to the development of envenomation treatments hold strong potential to provide rapid and high-throughput char- acterization of small molecule venom toxin inhibitors by determining their direct targets across diverse venom toxin protein families, accelerating identification of novel inhibitors. Here, we apply TPP and PISA methods to characterize the physical interactions between the SVMP inhibitor marimastat and toxin proteoforms of Crotalus atrox (Western Diamond- back Rattlesnake) venom. First, we determined toxin proteo- form presence and abundance in the venom of this well- studied species and used TPP to characterize the venom meltome by determining venom protein family-level and spe- cific proteoform-level thermal characteristics. Next, we per- formed PISA experiments within two different thermal windows to assess protein solubility changes upon inhibitor addition to identify specific proteoform targets of the small molecule inhibitor marimastat. Because of the previously characterized differences in signal-to-noise ratio between supernatant and pellet in PISA experiments (78, 79), we investigate and compare the targets identified in both the soluble and insoluble fractions. Our results demonstrate that a PISA-based approach can provide rapid, highly sensitive, and robust inferences for the unbiased proteome-wide screening of venom and inhibitor interactions. EXPERIMENTAL PROCEDURES Venom and Inhibitors C. atrox (Western Diamondback rattlesnake) venom was obtained by manual extraction from snakes housed at the University of Northern Colorado (UNC) Animal Facility, in accordance with UNC-IACUC protocols. Venoms were lyophilized and stored at −20 ◦C until use. Venoms were reconstituted at a concentration of 2 mg/ml and protein concentration was determined on a Nanodrop using the Absorbance 280 program. The small molecule matrix metalloprotease inhibitor marimastat ((2S,3R)-N4-[(1S)-2,2-Dimethyl-1-[(methylamino)carbonyl] propyl]-N1,2-dihydroxy-3-(2-methylpropyl)butanediamide, >98%, Cat no.: 2631, Tocris Bioscience) was reconstituted in ddH20 at a con- centration of 1.5 mM and stored at −20 ◦C. Venom Gland Transcriptomics An adult C. atrox (separate from those used for venom extraction) was collected in Portal, AZ under collecting permit 0456 and main- tained in the UNC Animal Facility. Four days following manual venom extraction, the C. atrox was humanely euthanized and venom glands removed (IACUC protocol no. 9204). Approximately 70 mg of tissue, originating from both left and right venom glands, was homogenized. Total RNA was isolated from homogenized venom gland tissue using the previously described TRIzol (Life Technologies) protocol for venom glands (80, 81). A NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs) was used to select mRNA from 1 μg of total RNA, and the NEBNext Ultra RNA library prep kit (New England Bio- labs) manufacture’s protocol followed to prepare the sample for Illu- mina RNA-seq. During library preparation, products within the 200 to 400 bp size range were selected by solid phase reversible immobili- zation with the Agencourt AMPure XP reagent (Beckman Coulter) and PCR amplification consisted of 12 cycles. Final quantification of the RNA-seq library was done with the Library Quantification Kit for Illumina platforms (KAPA Biosystems). The C. atrox venom gland RNA-seq library was then checked for proper fragment size selection and quality on an Agilent 2100 Bioanalyzer, equally pooled with eight other unique barcoded RNA-seq libraries and sequenced on 1/8th of an Illumina HiSeq 2000 platform lane at the UC Denver Genomics core to obtain 125 bp paired-end reads. To produce a comprehensive venom gland transcriptome database for C. atrox, two RNA-seq libraries were de novo assembled, the first from the C. atrox RNA-seq library detailed above and the second from a Texas locality C. atrox with reads available on the National Center for Biotechnology Information server (SRR3478367). Low quality reads were trimmed and adaptors removed using Trimmomatic (82) with a sliding window of four nucleotides and a threshold of phred 30. Reads were then assessed with FastQC (Babraham Institute Bioinformatics) to confirm that all adapters and low-quality reads were removed before de novo assembly. Three de novo assemblers were used in combination to produce a final, high-quality assembly: (i). first, a Trinity (release v2014-07-17) genome-guided assembly was completed us- ing default parameters and Bowtie2 (v2.2.6) (83) aligned reads to the C. atrox genome (provided by Noah Dowell (84)), (ii) a second de novo assembly was completed with the program Extender (k-mer size 100) (85), performed with the same parameters as used for other snake venom glands (86) and with merged paired-end reads, merged with PEAR (Paired-End read mergeR v0.9.6; default parameters) (87), as seed and extension inputs, and (iii) a third de novo assembly was completed with VT Builder using default settings (88). From a concatenated fasta file of all three assemblies, coding contigs were then identified with EvidentialGene (downloaded May 2018) (89) and redundant coding contigs and those less than 150 bps were removed with CD-HIT (90, 91). Reads were aligned with Bowtie2 to coding contigs and abundances determined with RNA-seq by Expectation- Maximization (RSEM; v1.2.23) (92). Contigs less than 1 transcript per million were filtered out, and the remaining contigs annotated with Diamond (93) BLASTx (E-value 10−05 cut-off) searches against the NCBI non-redundant protein database. Transcripts were identified as venom proteins after each was manually examined to determine if the resulting protein was full-length, shared sequence identity to a currently known venom protein, and contained a shared signal peptide sequence with other venom proteins within that superfamily. This transcript set was also filtered through ToxCodAn (94) as a final toxin annotation check, and the resulting translated toxins used as a custom database for mass spectrometry. Sequenced reads are available under NCBI accession SRR26583170, bioproject PRJNA1033617. These sequences were combined with those utilized in Calvete et al., (2009), and duplicate sequences removed. Venom Meltome Generation Thermal profiling assays were carried out following previously described methods (76, 95). Venom (1 μg/μl) was assayed in duplicate and divided into 10 aliquots of 20 μl and transferred to 0.2 ml PCR tubes. Each aliquot was individually heated at a single temperature over the range of 37 ◦C to 75 ◦C (37, 40, 45, 49, 52, 57, 62, 66, 69, 75 ◦C) for 3 min in a Bioer LifeECO thermal cycler (Fig. 1A). Samples were allowed to aggregate at 25 ◦C for 1 min and then placed on ice. Precipitated proteins were removed by centrifugation at 4 ◦C in 1.5 ml Eppendorf tubes (Thermo Fisher Scientific #3451) at 21,000g (14,000 RPM) for 45 min in an Eppendorf 5430 R centrifuge with an Eppendorf FA-45-30-11 rotor, and the supernatant was carefully removed with gel loading pipet tips (Fisher brand) and subjected to SDS-PAGE and sample preparation for mass spectrometric analysis. Inhibitor PISA Assays Venom (1 μg/μl) was incubated with two previously explored con- centrations of marimastat, 15 μM or 150 μM (53, 61), or a vehicle Mol Cell Proteomics (2024) 23(6) 100779 3 FIG. 1. TPP and PISA workflows. A, in TPP experiments, samples are heated between 40–70 ◦C and centrifuged to pellet denatured proteins. Samples are reduced, alkylated, and trypsin digested and analyzed with LC-MS/MS for protein identification. Melting curves are generated in ProSAP using unique intensity for each protein identified. Tm = melting temperature of 50% of population. B, in PISA, venom is incubated for 30 min at 37 ◦C with an inhibitor or alone. Samples are heated from 40 to 70 ◦C and pooled before centrifuging to pellet insoluble material. Samples are prepared as mentioned above and analyzed via LC-MS/MS for protein identification. To identify inhibitor targets, unique intensity is used to calculate SAR values for each protein followed by identification of significant outliers. PISA, proteome integral solubility alteration; TPP, thermal proteome profiling. Target Specificity of Marimastat Against Snake Venom Toxins control (ddH2O) for 30 min at 37 ◦C. Each sample was then divided into 12 aliquots of 20 μl in 0.2 ml PCR tubes. Each aliquot was indi- vidually heated at a different temperature from 40 ◦C to 70 ◦C for 3 min in a Bioer LifeECO Thermal cycler (Fig. 1B), allowed to cool at 25 ◦C for 1 min, and placed on ice. An equal volume of sample from each temperature point was pooled and centrifuged at 21,000g for 45 min at 4 ◦C to separate the soluble fraction from insoluble denatured proteins (70). Previous PISA performance assessment has demonstrated higher sensitivity in the precipitated fraction which showed a greater fold change in abundance compared to supernatant (Peng et al., 2016). Because of these previously characterized differences in performance between supernatant and pellet, we investigated both fractions (78, 79). The volume corresponding to 30 μg of soluble protein (based on controls) was taken from all samples (65) and prepared for mass spectrometry and 20 μg was used for gel electrophoresis. PISA assays for each condition were performed in triplicate. Because selection of a narrower temperature window for heat denaturation has been shown to increase sensitivity of the PISA assay (75), we also performed a temperature gradient denaturation with five temperatures (selected based on SVMP family-level Tm values) from 56 ◦C to 60 ◦C. Samples were pooled and processed as described above. High-Performance Liquid Chromatography One mg of venom incubated with either 150 μM marimastat or a vehicle control (ddH2O) was subjected to reverse phase high- performance liquid chromatography (HPLC) after heat treatment us- ing a Waters system, Empower software, and a Phenomenex Jupiter C18 (250 × 4.6 mm, 5 μm, 300 Å pore size) column as outlined in Smith and Mackessy (96). Proteins/peptides were detected at 280 nm and 220 nm with a Waters 2487 Dual λ Absorbance Detector. Fractions corresponding to each peak were collected and then frozen at −80 ◦C 4 Mol Cell Proteomics (2024) 23(6) 100779 overnight, lyophilized, and then separated with SDS-PAGE as previ- ously described (96). Percent peak area and peak height at 280 nm were recorded as a proxy for relative toxin abundance. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE materials were obtained from Life Technologies, Inc (Grand Island). DTT-reduced venom (20 μg) or lyophilized protein (approximately 5 μg—reverse-phase high-performance liquid chro- matography (RP-HPLC) fractionated) was loaded into wells of a NuPAGE Novex Bis-Tris 12% acrylamide Mini Gel and electro- phoresed in MES buffer under reducing conditions for 45 min at 175 V; 7 μl of Mark 12 standards were loaded for molecular weight estimates. Gels were stained overnight with gentle shaking in 0.1% Coomassie brilliant blue R-250 in 50% methanol and 20% acetic acid (v/v) and destained in 30% methanol, 7% glacial acetic acid (v/v) in water until background was sufficiently destained (approximately 2 h). Gels were then placed in storage solution (7% acetic acid, v/v) for several hours with gentle shaking at room temperature and imaged on an HP Scanjet 4570c scanner. Protein families were identified based on previously published reports and electrophoretic patterns for numerous rattlesnake venoms and several purified venom enzymes (97–99). Sample Preparation for Liquid Chromatography-Tandem Mass Spectrometry The volume of supernatant corresponding to 30 μg of venom pro- teins in the non-heat denatured control was dried in a vacuum centrifuge and redissolved in 8 M urea/0.1 M Tris (pH 8.5) and reduced with 5 mM tris (2-carboxyethyl) phosphine for 20 min at room tem- perature. Samples were then alkylated with 50 mM 2-chloroacetamide for 15 min in the dark at room temperature, diluted 4-fold with 100 mM Tris–HCl (pH 8.5), and trypsin digested at an enzyme/substrate ratio of Target Specificity of Marimastat Against Snake Venom Toxins 1:20 overnight at 37 ◦C. To stop the reaction, samples were acidified with formic acid (FA), and digested peptides were purified with Pierce C18 Spin Tips (Thermo Fisher Scientific #84850) according to the manufacturer's protocol. Samples were dried in a vacuum centrifuge and redissolved in 0.1% FA. Electrophoretic protein bands subjected to liquid chromatography- tandem mass spectrometry (LC-MS/MS) were excised from Coomassie-stained gels, destained, and subjected to in-gel reduction, alkylation, and overnight trypsin digestion as previously described (100). Following the overnight digestion, samples were acidified with 5% FA and tryptic peptides were extracted in 30 μl of 50% acetoni- trile/1% FA. Digests were dried in a vacuum centrifuge and redis- solved in 0.1% FA for mass spectrometry. Nano Liquid Chromatography Tandem Mass Spectrometry Nano liquid chromatography tandem mass spectrometry was per- formed using an Easy nLC 1000 instrument coupled with a Q-Exactive HF Mass Spectrometer (both from ThermoFisher Scientific). Approxi- mately 3 μg of digested peptides were loaded on a C18 column (100 μm inner diameter× 20 cm) packed in-house with 2.7 μm Cortecs C18 resin and separated at a flow rate of 0.4 μl/min with solution A (0.1% FA) and solution B (0.1% FA in ACN) under the following con- ditions: isocratic at 4% B for 3 min, followed by 4 to 32% B for 102 min, 32 to 55% B for 5 min, 55 to 95% B for 1 min and isocratic at 95% B for 9 min. Mass spectrometry was performed in data- dependent acquisition mode. Full MS scans were obtained from m/z 300 to 1800 at a resolution of 60,000, an automatic gain control target of 1 × 106, and a maximum injection time of 50 ms. The top 15 most abundant ions with an intensity threshold of 9.1 × 103 were selected for MS/MS acquisition at a 15,000 resolution, 1 × 105 automatic gain control, and a maximal injection time of 110 ms. The isolation window was set to 2.0 m/z and ions were fragmented at a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Analysis of Mass Spectrometry Data Fragmentation spectra were interpreted against a custom protein sequence database, comprising 570 entries generated from the as- sembly of C. atrox venom gland transcriptome data (described above) combined with UniProt entries of all toxins found in the C. atrox venom proteome (97), reverse decoys and contaminants, and using MSFragger v3.8 within the FragPipe (v20.0) computational platform (101, 102). Cysteine carbamidomethylation was selected as a fixed modification, oxidation of methionine was selected as a variable modification, and precursor-ion mass tolerance and fragment-ion mass tolerance were set at 20 ppm and 0.4 Da, respectively. Fully tryptic peptides with a maximum of two missed tryptic cleavages were allowed and the protein-level and peptide-level false discovery rate was set to <1%. The relative abundance of major snake venom toxin families was compared across samples using sum-normalized total spectral intensity (103). Analysis of TPP Data Protein melting curves were generated by fitting sigmoidal curves to relative protein abundances using the Protein Stability Analysis Pod (ProSAP) package (104). The temperature at which relative protein abundance reached 50%, Tm (melting temperature), was determined in ProSAP by normalizing intensity to the lowest temperature (37 ◦C), followed by normalization to the most thermostable proteins as pre- viously described (79). Duplicates were averaged to determine the average Tm of all identified venom toxins. Venom proteins failing to reach 50% denaturation even at higher temperatures were classified as non-melting proteins. Analysis of PISA Data PISA data was analyzed as previously described (71, 103). Briefly, PISA uses the ΔSm value or soluble abundance ratio (SAR) as opposed to the Tm to determine differences in thermal stability (70). ΔSm repre- sents the difference in integral abundance of a protein in treated compared to untreated samples.We performed a two-tailed Student’s t test with unequal variance to calculate p-values (p < 0.05). Changes to venom protein abundance were visualized using volcano plots based on log2SAR values and -log10 transformed p-values. Proteins with a log2SARvalue≥0.5 or≤−0.5 anda -log10 transformedp-value≥1.3 (p< 0.05) were identified as toxins with a significant shift in solubility. All figures were made with BioRender.com. RESULTS C. atrox Venom Proteome Previous characterization of the C. atrox venom proteome revealed the presence of at least 24 proteins belonging to eight different venom toxin protein families (Fig. 2A; (97)) and, more recently, the presence of 31 SVMP genes in C. atrox with 15 to 16 expressed SVMPs (105). SVMPs and snake venom serine proteases (SVSPs)were the twomost abundant protein families representing nearly 70% of the venom proteome. L-amino acid oxidase (L-AAO), phospholipase A2 (PLA2), disintegrins, and cysteine-rich secretory proteins (CRISPs) comprise most of the remaining 25%ofC. atrox venom proteins, whereas vasoactive peptides, endogenous SVMP inhibitors, and C-type lectins (CTLs) comprised the remaining small fraction of venom com- ponents comprising <2% of the venom proteome. Utilizing the protein database generated fromprotein sequences of proteins identified by Calvete et al. (97) combined with protein se- quences derived from a C. atrox venom gland transcriptome, we detected 46 unique proteoforms with at least one unique peptide in C. atrox venom (Fig. 2B). Venom toxins with the highest number of distinct proteoforms detected included 13 CTLs, 13 SVMPs, nine SVSPs, and three PLA2s (Fig. 2B). We identified only one unique proteoform of more abundant pro- teins including L-AAO and CRISP and only one proteoform for minor components bradykinin-potentiating peptide (BPP), glutaminyl-peptide cyclotransferase, hyaluronidase, nerve growth factor, phospholipase B, and vascular endothelial growth factor (VEGF). RP-HPLC analysis revealed a complex toxin profile ofC. atrox venom similar to that of Calvete et al., 2009 (Fig. 2C). We used SDS-PAGE of peak fractions (Supplemental Fig. S1) combined with the peak elution times and identifiedmasses inCalvete et al. (97) to confirm peak identities. BPP’s eluted between 8 and 15min with co-elution of disintegrins and SVMP inhibitors. PLA2 eluted at 27, 30, and between 39 to 44 min, CRISP eluted at 29 min, SVSP eluted between 32 to 36 min, L-AAO eluted at 46 min, and SVMPs eluted between 51 to 56 min (Fig. 2C). C. atrox Venom Meltome With the goal of demonstrating the utility of applying a PISA workflow for identifying venom protein interactions with Mol Cell Proteomics (2024) 23(6) 100779 5 http://BioRender.com FIG. 2. Crotalus atrox venom proteome characterization. A, toxin family abundances in C. atrox venom modified from Calvete et al., 2009. B, the number of proteoforms identified in C. atrox venom in the present study organized by family. C, RP-HPLC separated C. atrox venom. For peak identification, fractions were analyzed with mass spectrometry and SDS-PAGE and compared to known masses from Calvete et al., 2009. PP, bradykinin potentiating peptide; CRISP, cysteine rich secretory protein; CTL, C-type lectin; Dis, disintegrin; GPC, glutaminyl-peptide cyclotransferase; HPLC, high-performance liquid chromatography; HYAL, hyaluronidase; L-AAO, L-amino acid oxidase; NGF, nerve growth factor; PLA2, phospholipase A2; PLB, phospholipase B; RP-HPLC, reverse-phase high-performance liquid chromatography; SVMP, snake venom metalloprotease; SVMPi, SVMP tripeptide inhibitor; SVSP, snake venom serine protease; VEGF, vascular endothelial growth factor. Target Specificity of Marimastat Against Snake Venom Toxins a small molecule inhibitor, we first assessed the effects of thermal stress on the venom proteome. Venom was sub- jected to increasing temperatures ranging from 40 to 75 ◦C, allowed to cool at room temperature, followed by separation and removal of aggregates from each temperature point by centrifugation. The soluble fractions were then visualized by gel electrophoresis (Fig. 3A) and prepared for LC-MS/MS. SDS-PAGE analyses of these fractions indicate that the entire venom proteome appeared to exhibit some degree of denaturation between the temperatures tested with clear differences in denaturation observed across venom protein families (Fig. 3A). For example, L-AAO, hyaluronidase, SVMPs, and CTLs appeared more thermally sensitive while SVSP, CRISP, PLA2, and disintegrin families exhibited greater stability at higher temperatures. Next, we assessed the thermal stability of venom proteins across the ten different temperature points by LC-MS/MS. 6 Mol Cell Proteomics (2024) 23(6) 100779 An equal volume of each soluble fraction was collected and subjected to reduction, alkylation, trypsin digestion, and LC-MS/MS. Fragmentation spectra were interpreted against our C. atrox–specific custom venom proteome sequence database, and we used the ProSAP package (104) to determine melting points for each venom protein family. When normalized to thermostable proteins, most venom proteins show decreasing abundance with increasing tem- perature, with the majority of proteins reduced in abundance at temperatures above 62 ◦C (Fig. 3B). The distribution of toxin melting temperature (Tm) values ranged from 47.8 to 74.3 ◦C (Fig. 3, C and D; Supplemental Table S1). Most toxins had Tm’s between 50 to 60 ◦C (Fig. 3D; n = 22) or 60 to 70 ◦C (n = 28), and only 11 proteoforms were still ther- mostable with no calculable Tm at 75 ◦C (BIP, BPP, VEGF, 4 PIII-SVMPs, and 4 SVSPs; Fig. 3D). The five toxins with the lowest Tm’s included four CTLs (average Tm = 49.3 ◦C, A C D E F G B FIG. 3. Crotalus atrox meltome characterization. A, SDS-PAGE of C. atrox venom heated at temperatures between 37–70 ◦C for 3 min. B, heatmap showing the soluble fraction of most toxin proteoforms relative to 37 ◦C in C. atrox venom at 40, 45, 49, 52, 57, 62, 66, 69, and 75 ◦C. Heatmap colors represent the number of SDs away from the mean of the protein’s intensity in each row of the heatmap. C, distribution of melting temperatures organized by family. Dotted lines represent median and quartile ranges. D, distribution of melting temperatures for all toxins identified. Nonmelters (NM) are classified as proteoforms for which Tm could not be calculated when heated to a maximum temperature of 75 ◦C. E, representative melting curve of a CTL (Crotocetin). Abundance is normalized to 37 ◦C. F, representative melting curve of a PLA2 (Cvv-N6). Abundance is normalized to 37 ◦C. G, representative melting curve of an SVMP (PIII 28325). Abundance is normalized to 37 ◦C. CTL, c-type lectin; SVMP, snake venom metalloprotease. Target Specificity of Marimastat Against Snake Venom Toxins stdev = 1.1 ◦C; Fig. 3, C and E) and the single hyaluronidase proteoform (Tm = 50.6 ◦C). CTLs Tm values as a whole ranged from 47.8 to 63.1 ◦C (ave = 56.0 ◦C, stdev = 5.3 ◦C). PLA2s had an average Tm of 61.2 ◦C (stdev = 8.3 ◦C; Fig. 3, C and F). The different SVMP subfamilies differed slightly in their melting range but were not significantly different (p = 0.77; Fig. 3C). PI-SVMP proteoforms had an average Tm of 56.8 ◦C (stdev = 0.03 ◦C), PIIs averaged 59.3 ◦C (stdev = 5.7 ◦C; Fig. 3C), and PIII’s averaged 59.7 ◦C (stdev = 6 ◦C; Fig. 3G). SVSPs had the highest average Tm (63.9 ◦C, stdev = 5.5 ◦C) and made up a large proportion of the proteins that were thermostable above 75 ◦C (Fig. 3B). The single L-AAO proteoform identified melted at 61.7 ◦C. In general, melting temperatures were reproducible between replicates with an average SD of 1.17 ◦C between repli- cates. These results demonstrate protein family-level dif- ferences in thermal stability, in that all proteoforms of some families denatured (i.e., CTL, SVMP I) when subjected to heat, while others appear resistant to thermal perturbation (SVSPs). These results indicate that a significant proportion of the venom proteome is amenable to thermal denaturation. Mol Cell Proteomics (2024) 23(6) 100779 7 Target Specificity of Marimastat Against Snake Venom Toxins Venom-Wide Interactions with Marimastat After establishing that venom proteins are susceptible to thermal denaturation, we next assessed if a PISA strategy could be applied to elucidate small molecule-venom protein engage- ment. For this, we applied the PISA assay, an approach where samples across the entire temperature gradient of the same treatment are pooled prior to preparation and mass spectro- metric analysis (66, 70, 71).With traditional PISA, the abundance of the protein(s) in the soluble fractions of the pooled samples is then used to assess the effect of a compound on its solubility (70). For highly thermostable proteins, monitoring supernatant alone is likely not effective in thermal-shift–based methods (79). Because increases in protein solubility upon compound binding also result in decreased protein abundance within the precipi- tated pellet, quantifying protein abundance in the precipitate pellet can also identify protein targets (79). Further, because of different observed signal-to-noise ratios, soluble and pelleted materials may perform differently in PISA assays to identify sig- nificant thermal shifts (78, 79). Utilizing precipitated material to measure changes in protein solubility can additionally reduce the FIG. 4. Crotalus atrox venom-wide interactions with two concentra volcano plot comparing soluble supernatant of heat-treated venom + proteoforms, red = positive outliers, blue = negative outliers, gray = no treated venom + marimastat (150 μM) to heat-treated venom alone. C, marimastat (15 μM) to heat-treated venom alone. D, volcano plot compar to heat-treated venom alone. SVMP, snake venom metalloprotease. 8 Mol Cell Proteomics (2024) 23(6) 100779 false discovery rate and improve sensitivity of the assay. Based on this logic, we utilized both supernatant and precipitated ma- terial to investigate the effects of marimastat. PISA assays were performed on marimastat, an inhibitor of matrix metalloproteases that has shown significant inhibitory activity against SVMPs (50, 51, 53, 56, 60–62). C. atrox venom was incubated with marimastat (15 μM and 150 μM) or vehicle (ddH2O) for 30 min at 37 ◦C. Following incubation, each sample was divided into 12 aliquots and subjected to increasing temperatures from 40 to 70 ◦C. Equal aliquots per temperature point were then pooled, protein aggregates were separated by centrifugation, and the soluble and insoluble fractions of the vehicle and inhibitor-treated venoms were prepared for downstream analysis. When filtering criteria were applied (p < 0.05, and log2- SAR>0.5), the lower concentration of marimastat (15 μM) caused five of 21 SVMP proteoforms (PIII 28348, PIII 28325, PII 25887, PII 23541, and VAP 1) to display a stabilizing shift in treated supernatant compared to untreated supernatant (Fig. 4A). In addition to these five proteoforms, two additional tions of marimastat with temperature window from 40 to 70 ◦C. A, marimastat (15 μM) to heat-treated venom alone. X indicates SVMP t significant. B, volcano plot comparing soluble supernatant of heat- volcano plot comparing insoluble precipitate of heat-treated venom + ing insoluble precipitate of heat-treated venom + marimastat (150 μM) Target Specificity of Marimastat Against Snake Venom Toxins SVMPs (PII 23556 and PII 27392) were more abundant in the supernatant of venom treated with 150 μM of marimastat (Fig. 4B; Supplemental Table S2). Next, we compared the pellets of untreated venom to venom treated with both concentrations of marimastat. When filtering criteria were applied at the low concentration, only four SVMPs (Atro B, Atro-D, SVMPIII 27520, and SVMPIII 28348) were detected at significantly lower abundance in the treated pellet than the control pellet, indicative of a stabilizing effect of marimastat (Fig. 4C). These same proteoforms, in addition to SVMP PII 23541, were also significantly reduced in the pellet of the higher marimastat concentration (Fig. 4D). The presence of positive outliers identified in both the supernatant and negative outliers in the precipitate of treated venom in- dicates an overall stabilizing effect of marimastat on venom targets. Validation of Inhibitor Interactions To validate our PISA results showing the stabilizing effects of marimastat on SVMPs, we performed SDS-PAGE and RP- HPLC on non-heat-denatured venom and venoms treated FIG. 5. Validation assays of inhibitor interactions. A, SDS-PAGE com 150 μM of marimastat with a thermal window of 40–70 ◦C. Note the recov treated samples. The left side indicates molecular mass standards in abundance of non-heat-treated venom (black), heat-treated venom (red) (blue). Note the recovery of peak area in the inhibitor-treated sample. C, e non-heat-treated venom (black), heat-treated venom (red), and venom he recovery of PLA2 peak area in the inhibitor-treated sample. HPLC metalloprotease. with marimastat or vehicle. The toxin family composition of the peaks and bands altered by the addition of marimastat was confirmed by mass spectrometry (Supplemental Tables S3 and S4). In the heated marimastat-treated venom, gel bands A and B were composed predominantly of PIII 27501 and VAP2B (band A) and PII 23556, Atro E and Atro B (band B; Fig. 5A). Band C was composed of acidic PLA2, band D of PLA2 Cax-K49, CTL 22443, and PLA2 Cvv-N6, and band E was predominantly CTL 21182, CTL 22444, and PLA2 Cax- K49. SDS-PAGE analysis of the heat-denatured and unde- natured control venom shows a clear reduction in the size and intensity of SVMP-PIII (~50 kDa; band A), SVMP P-I/II (~20 kDa; band B), and CTL/PLA2 gel bands (~10–14 kDa; bands C-E) in response to thermal treatment (Fig. 5A). This reduction in SVMP and CTL/PLA2 band size and intensity in response to heat appears to be partially to fully recovered when venom is incubated with 150 μM marimastat (Fig. 5A). RP-HPLC peaks eluting between 51 to 54 min were iden- tified by mass spectrometry as SVMPs, with VAP2B, PIII 27501, P-III ACLD, PII 23556, PIII 28348, PII 23566, and Atro E representing the dominant proteoforms (Fig. 5B). These parison of heat-treated venom to heat-treated venoms incubated with ery in band size and intensity of SVMP and PLA2 bands in marimastat- kDa. B, enlarged HPLC-separated SVMP peak overlay comparing , and venom heat-treated after incubation with 150 μM of marimastat nlarged HPLC-separated PLA2 peak overlay comparing abundance of at-treated after incubation with 150 μM of marimastat (blue). Note the , high-performance liquid chromatography; SVMP, snake venom Mol Cell Proteomics (2024) 23(6) 100779 9 Target Specificity of Marimastat Against Snake Venom Toxins continued to be the dominant proteoforms with the exception of Atro E in both the heated control and marimastat-treated venoms. The 27-min, 29-min, and 30-min peaks were composed predominantly of the basic PLA2 Cax-K49, CRISP, and basic PLA2 Cvv-N6, respectively (Fig. 5C). These remained the dominant proteoforms in the heated control venom and marimastat-treated venom, with the exception of marimastat-treated peak 30 where CRISP became the domi- nant proteoform followed by PLA2 Cvv-N6. The stabilizing effect of marimastat on some venom pro- teins is further demonstrated by the analysis of RP-HPLC, which shows partial recovery in the chromatographic peak area and peak height of SVMP and two PLA2 peaks in the marimastat-treated venoms compared to the controls (Fig. 5, B and C). After heat treatment, SVMPs lose 44% of their original peak area, but marimastat treatment results in only a 7% decrease in peak area after melting (Fig. 5B). The PLA2 proteins eluting at 27 min decreases by 75% when venom is heat-treated and only 26% when venom is treated with mar- imastat, while the PLA2 eluting at 30 min is virtually absent in the heated control venom but only loses 52% abundance when heat-treated with marimastat (Fig. 5C). Peak heights of PLA2 (27 min), PLA2 (30 min), and SVMPs decrease by 81%, 100%, and 57% respectively after melting; however, with marimastat, peak height only decreases by 21%, 48%, and 21%, respectively (Fig. 5, B and C). VAP2B is the dominant proteoform in C. atrox venom (Fig. 5B; Supplemental Tables S3 and S4) and was the second most abundant proteoform in the SVMP fractions and gel bands of treated venom. However, it was not detected as a stabilized outlier in either supernatant or pellet in the current PISA experiments performed with the temperate range of 40 to 70 ◦C. Thus, we aimed to increase the sensitivity of the PISA assay with a narrower thermal window determined by the Tm values previously calculated for the target toxin family. Venom-Wide Interactions with Marimastat in a Narrowed Thermal Window PISA While PISA is advantageous because it reduces the analysis time and sample preparation while still being effective at target discovery, it may sacrifice sensitivity compared to TPP ex- periments due to the pooling of all temperature points, and melting temperature selection can have a drastic effect on thermal behavior in PISA experiments (75, 79). To investigate this, we compared the performance of a broad thermal win- dow (40–70 ◦C) to a narrower window (56–60 ◦C), selected based on the mean and SD of Tm’s of SVMPs, the target venom toxin family. We performed PISA assays with the same concentrations of marimastat, with a narrower temperature window from 56 to 60 ◦C with five temperature points of each sample replicate, which has been shown to improve the overall sensitivity of the PISA assay in target identification (75). When samples were heat treated with a narrower window of temperatures, the lower concentration of marimastat 10 Mol Cell Proteomics (2024) 23(6) 100779 displayed three of 21 SVMP proteoforms (PIII 28348, PIII 28325, PII 23541) at a greater abundance in treated super- natant than untreated supernatant (Fig. 6A; Supplemental Table S4). At the higher concentration, seven of 21 proteo- forms were higher in supernatant of treated venom: VAP1, PIII 28348, PIII 28325, PII 23556, PII 23541, PIII 28771, PIII 27392 (Fig. 6B). In the pellets of samples treated with a narrower range of temperatures, 11 of 21 proteoforms (VAP2B, PIII 28348, Atro D, PIII 27501, PII 23541, Atro B, PIII 27461, PIII 27520, PIII 28771, PII 23648, PIII 27392) demonstrated an increase in solubility in the pellet of the lower concentration of marimastat condition (Fig. 6C). These same proteoforms plus PII 23556 were reduced in the pellet at the higher concen- tration of marimastat (Fig. 6D). Heat-Treatment Comparison Next, we compared the performance of a broad thermal window (40–70 ◦C) and a narrower thermal window (56–60 ◦C) to the results gathered from our validation experiments (Figs. 4–6). At both temperature ranges when venom was treated with marimastat, principal component analysis shows clustering of the replicates based on treatment condition, with the two marimastat-treated groups separating from the vehicle-treated samples (Fig. 7, A–D). These results indicate that both concentrations of marimastat interact with venom protein targets and alter the solubility of venom proteins compared to the control group. However, pellet replicates (Fig. 7, B and D) cluster more tightly together in both condi- tions than in supernatants with greater separation among the treatment groups (Fig. 7, A and C). The highest amount of variance explained (97%) by the top two principal components was in the narrow window pellet, though all plots had a high percentage of sample variance explained (>86%). The number of significantly stabilized proteins found in the supernatant (p < 0.05, log2SAR ≥0.5) after treatment with marimastat at a broad melting window were 12 and 14 for 15 μM and 150 μM, respectively (Fig. 7E). The percentage of SVMPs among the identified proteins was 42% and 50%. At the narrower melting window, four and nine proteins were identified in 15 μM and 150 μM treatments, respectively, but SVMPs comprised 75% and 78% of identified proteins. In general, pellets of both melting windows appeared to perform better regardless of concentration. The precipitated pellet from 150 μM-treated venom heated at the narrower thermal window identified the most SVMP proteoforms of any treatment group. Though the broader temperature window precipitate identified fewer SVMP proteoforms, SVMPs were the only venom toxin family proteoforms identified, while the supernatant appeared to contain more, potentially off-target, non-SVMP identifications. The broad melting window identified four and five SVMP proteoforms, while the narrow window pellets identified 11 and 12 for 15 μM and 150 μM, respectively (Fig. 7E). Performance of specific SVMP proteoform identification between melting windows varied significantly at both FIG. 6. Crotalus atrox venom-wide interactions with two concentrations of marimastat with temperature window from 56 to 60 ◦C. A, volcano plot comparing soluble supernatant of heat-treated venom + marimastat (15 μM) to heat-treated venom alone. X indicates SVMP proteoforms, red = positive outliers, blue = negative outliers, gray = not significant. B, volcano plot comparing soluble supernatant of heat- treated venom + marimastat (150 μM) to heat-treated venom alone. C, volcano plot comparing insoluble precipitate of heat-treated venom + marimastat (15 μM) to heat-treated venom alone. D, volcano plot comparing insoluble precipitate of heat-treated venom + marimastat (150 μM) to heat-treated venom alone. SVMP, snake venom metalloprotease. Target Specificity of Marimastat Against Snake Venom Toxins concentrations, with only one common proteoform at 15 μM (Fig. 7F) and two at 150 μM (Fig. 7G). At both concentrations, the narrow window pellets had the highest number of uniquely identified SVMP proteoforms. When soluble abundance ratios (log2SAR) of supernatant and pellets are compared, the nar- rower thermal window performs significantly better at identi- fying the target and off-target proteoforms that meet significance criteria in both supernatant and pellet. When only proteins meeting significance criteria for both supernatant and pellet were compared, the broad window performed poorly, identifying only two SVMPs (PIII 28348, PII 23541) with sig- nificant stabilizing shifts (Fig. 7H). The narrower window identified six SVMP proteoforms with significant stabilizing shifts (VAP1, PII 23556, PIII 28348, PIII 28771, PII 23541, PIII 27392) (Fig. 7I). Off-target proteins that met significance criteria for both conditions included hyaluronidase and, using the narrower window, three SVSPs and VEGF. Coefficients of Variance To determine consistency of label-free quantification mea- surements and ensure that observed alterations are reliable, coefficients of variance (CVs) were calculated for each venom protein across the different replicates in each group. These individual protein CVs were then averaged to determine the mean variance for each analysis group. In general, CVs were low across replicate groups, with an average intra-group CV of 17.2% for all venom proteins, including all analysis groups and both narrow and wide melt range data. Average intra-group CVs were lowest for the high marimastat supernatant (10.33%), high marimastat pellet (10.68%), and low marima- stat pellet (13.69%) groups. Average CVs were greater for the low marimastat supernatant (26.61%), control supernatant (18.57%), and control pellet (23.33%) groups. However, all comparisons used to draw conclusions maintain significance despite this variability. SVMP Comparisons and Target Identification Because the narrower temperature window appears to identify more target proteoforms with less noise, we utilized this narrow window approach to re-analyze interactions be- tween SVMPs and marimastat. More SVMP proteoforms were identified as significant with a narrower window when Mol Cell Proteomics (2024) 23(6) 100779 11 A E B F H I G C D FIG. 7. Comparison of a broad (40–70 ◦C) to a narrow (56–60 ◦C) PISA thermal window. PCA plot comparing replicates of soluble su- pernatant of (A) heat-treated venom + 15 μM marimastat to heat-treated venom alone and (B) heat-treated venom +150 μM marimastat to heat- treated venom alone. PCA plot with 95% confidence intervals comparing replicates of insoluble precipitate of (C) heat-treated venom +15 μM marimastat to heat-treated venom alone and (D) heat-treated venom +150 μM marimastat to heat-treated venom alone. E, number of SVMP and non-SVMP proteins identified with significant thermal shifts toward stabilization (p-value < 0.05 log2SAR> 0.5) after 15 μM or 150 μM marimastat treatment in supernatants and pellets heat-treated at a broad (40–70 ◦C) or a narrow (56–60 ◦C) thermal window. Venn diagrams of SVMP proteins identified with significant thermal shifts toward stabilization (p-value < 0.05, log2SAR > 0.5) in supernatants and pellets heat-treated at a broad (40–70 ◦C) or a narrow (56–60 ◦C) thermal window after (F) 15 μM or (G) 150 μM marimastat treatment. Scatter plot showing log2SAR Target Specificity of Marimastat Against Snake Venom Toxins 12 Mol Cell Proteomics (2024) 23(6) 100779 Target Specificity of Marimastat Against Snake Venom Toxins analyses of the supernatant and pellet were combined (Fig. 7, E–G), and analyses of the pellet identified more SVMP pro- teoforms than the supernatant within the narrower thermal window (Fig. 8A). Specifically, analysis of the narrow-range pellet identified highly abundant proteins also identified in the validation assays but not identified using the broad ther- mal window (e.g., VAP2B). Hierarchical clustering analysis comparing supernatants and pellets at both concentrations shows an inverse relationship between relative intensity of each proteoform in the pellet versus the supernatant (Fig. 8B). When these conditions are compared to controls, we resolved three patterns of various proteoforms: (1) proteoforms that showed a positive shift (trend towards stabilization) in the supernatant at both concentrations of marimastat; (2) pro- teoforms that disappear from the pellet after marimastat treatment but do not necessarily increase in SAR in the su- pernatant (trend towards stabilization); and (3) proteoforms that increase in pellet SAR after treatment (Fig. 8C). Finally, correlation analysis performed with the SAR values of pro- teoforms supernatants and pellets of the narrow thermal window identified three clusters of proteoforms with similar shifts in thermal behavior: (1) a cluster containing the pro- teoforms that were stabilized by marimastat (e.g., VAP2B, 27501, 23556, 27392) that represent the strongest targets of marimastat, (2) a cluster containing atrolysin B, atrolysin D, and PIII 27520 which appeared to decrease in abundance in both supernatants and pellets after marimastat treatment, and (3) a cluster that did not appear to be thermally stabilized by marimastat including atrolysin A, PII 24293 (Fig. 8D). The strongest target list includes proteoforms identified in the validation protein gel (e.g., VAP2B, PIII 27501, PII 23556, PIII 28348, PIII 27392, and PII 23648) and those identified as most abundant in the SVMP HPLC peaks (VAP2B, PIII 27501, PII 23556, PIII 28348, PIII 27461). While the most abundant proteoforms were identified as significant in analysis of the pellet but not in the corresponding supernatant, there does appear to be more noise in the pellet data indicated by the number of non-target proteins that reached significance with a p < 0.05, log2SAR≥ 0.5 (upright right quadrants; Figs. 4, C and D and 6, C and D). This could be due to differences in solubilization of proteins in the pellet indicating that the soluble fraction analysis is more reliable in quantification. However, to examine the validity of analyzing the pellet for identifying high abundance targets, we use VAP2B as an example. In the case of VAP2B, the stabilizing effect of marimastat is only evident in the pellet, and both 15 μM and 150 μM concentrations have significantly lower levels of the precipitated toxin (p = 0.003, p = 0.0013, respectively, Fig. 8E). A less abundant proteoform, PIII 28348, values calculated from 150 μM marimastat-treated venom versus vehic significance criteria in both supernatant (SN) and precipitate (Pellet) grou ACLD, PIII SVMP ACLD; black, SVMP proteoforms; Con, control; CTL, C- component analysis; Pell, pellet; PISA, proteome integral solubility altera showed an increase in solubility that was detected in both the pellet and the supernatant at both concentrations of mar- imastat (Fig. 8F). In the pellet, abundance of PIII 28348 was significantly lower at both concentrations than in the untreated control (p = 0.0004, p = 0.0014, respectively) and significantly higher in the supernatant compared to control (p < 0.0001). Off-Target Effects Based on recovery of peak area in PLA2 and CTL-containing peaks and gel bands in marimastat-treated venom subjected to RP-HPLC (Fig. 5C), we explored the possibility of using PISA assays to detect off-target effects of marimastat. Inter- estingly, at both concentrations with the wider thermal win- dow, we observed changes in thermal behavior indicative of a shift towards stabilization of some non-target protein families, including two PLA2 proteoforms (Cax-K49 and Cvv-N6) and four CTL proteoforms. In the gels, there was recovery of band E containing Cax-K49 and CTL’s 22443, 21182, and 22444. Both PLA2 proteoforms were repeated positive outliers in supernatants of all conditions but were not significant in any pellets. Various CTL proteoforms including CTL 21107, 22105, 22447, 21150, and 22232 were significant outliers in some conditions. When comparing only significant log2SAR values in both supernatant and pellet, hyaluronidase, VEGF, two SVSPs, and two CTL’s (22444, 21107) were significantly correlated between pellet and supernatant at the narrower melt window (Fig. 7I). DISCUSSION The development and testing of alternative snakebite ther- apeutics that are affordable, stable, and easily administered is an urgent global need (1, 10, 47, 106). Small molecule in- hibitors currently lead the field of possible supplementary snake envenomation therapies, with phase II clinical trials ongoing for the PLA2 inhibitor varespladib (107) and the SVMP inhibitor DMPS ((108); ClinicalTrials.gov, 2021). Numerous in- hibitors have shown promising cross-species efficacy in vivo and in vitro (37, 50, 53, 57, 109), indicating that they may be less vulnerable to the effects of venom variation than tradi- tional antibody-based antivenoms. However, additional pre- clinical studies are needed to evaluate the neutralizing efficacy and specificity of these drugs alone and in combination, and the development of these drugs would be accelerated by implementation of high throughput screening of interactions and efficacy across many species. Research on small mole- cule inhibitors of snake venom toxins has typically focused on in vitro and in vivo functional assays based on the known or likely biological activities of toxins (49–53, 58, 109–113). These le treatment heated at from (H) 40–70 ◦C or (I) 56 to 60 ◦C that meet p. Largest outliers of SVMP and non-SVMP proteoforms are labeled. type lectin; gray, non-SVMP toxins; Hyal, hyaluronidase; PCA, principal tion; SN, supernatant; SVMP, snake venom metalloprotease. Mol Cell Proteomics (2024) 23(6) 100779 13 http://ClinicalTrials.gov A C D E F B FIG. 8. Effects of marimastat treatment and a narrow thermal window on SVMP proteoforms only. A, number of proteins identified in supernatant (SN) and pellet of narrow thermal window that meet significance criteria (p-value < 0.01, log2SAR> 0.5); (B) heatmap of sum- normalized intensity values in supernatant or precipitate of SVMPs from 15 μM or 150 μM marimastat-treated venom. Heatmap colors are Target Specificity of Marimastat Against Snake Venom Toxins 14 Mol Cell Proteomics (2024) 23(6) 100779 Target Specificity of Marimastat Against Snake Venom Toxins approaches utilize a downstream measurement of the pre- sumed interactions of an inhibitor with its targets (ex. reduced specific activity or increased survival). A previous study per- formed molecular docking analysis using marimastat and a purified PI SVMP proteoform CAMP-2 to demonstrate a direct interaction (51). However, the PISA method outlined here represents both a direct and venom-wide assessment of target-ligand engagement and provides the opportunity to link direct target–ligand interactions with functional and pheno- typic responses (71, 72, 114). In this study, we investigate the thermal characteristics of the C. atrox venom proteome and use this to develop a PISA- based assessment of the venom proteome-wide targets of the SVMP inhibitor marimastat. We investigate both its proteome- wide effects and determine and validate its interactions with specific venom proteoforms of its target toxin family (SVMPs) as well as possible off-target protein families. We identified a suite of marimastat proteoform-level targets and confirmed them by RP-HPLC and SDS-PAGE. We also compared the performance of soluble supernatant and insoluble precipitate at two different inhibitor concentrations for target identifica- tion. Our results provide a promising first assessment of the application of a PISA-based approach as a sensitive and high- throughput method to assess the direct targets of small molecule inhibitors for snake venom. Based on our experi- ments with PISA in this context, we find that analysis of the insoluble fraction from venom that was treated with a high concentration of marimastat, but a narrow thermal window for PISA, provided more sensitive target data with the least noise than other tested methods. Previous research has shown that small molecule inhibitor efficacy in vitro may not always translate to in vivo efficacy. For example, the SVMP inhibitor prinomastat and the metal chelator dimercaprol showed moderate to high SVMP inhibi- tory activity in vitro but failed to confer any protection towards crude venom in in vivo assays (60). Furthermore, studies have highlighted cross-species variation in neutralization effects of potential inhibitors, which has significant implications for the application of inhibitors as broadly effective pre-hospital treatments of envenomation by potentially diverse species (58). Dimercaprol showed promise in murine models as an SVMP inhibitor against Echis ocellatus venom (115); however, it lacked this protective effect in vivo against Dispholidus typus venom (60), likely due to the high levels of divergence in venom composition between these distantly related species. While some inhibitors have demonstrated neutralization scaled by row to better visualize variation in sum-normalized intensity supernatant or precipitate of SVMPs from 15 μM or 150 μM marimasta shifts in abundance. Heatmap colors are scaled by row to better visual tion plot of SAR values showing strongest marimastat targets based o Comparison of concentration-dependent intensity shifts between prec VAP2B and (F) a less abundant SVMP proteoform PIII 28348 at both con venom metalloprotease. capacity of specific biological effects (such as anticoagulation) caused by venoms of divergent species, they may vary in effectiveness across species because of lineage-specific variation in venom toxin sequence, activity, or relative abun- dance or because the same biological effects may arise due to the action of different toxin families altogether (58, 60). Knowledge of the snake species-specific venom-wide and proteoform-specific efficacy of inhibitors has the potential to significantly improve our ability to predict cross-species neutralization and to unravel the disparity between in vitro and in vivo results. Before PISA could be widely applied for the screening of a large number of potential inhibitors against snake venom, a number of considerations must be addressed. By pooling a wide range of temperature points, PISA data in particular may suffer from reduced screening sensitivity, depending on the specific thermal properties of various proteins (75). Venom toxins appear to have significantly higher Tm values than hu- man cell types, which ranged from 48 to 52 ◦C (95). Some potential snake venom toxin families of interest (i.e., SVSPs and CRISPs) display high thermal tolerance likely due to increased frequency of disulfide bonds and other post- translational modifications, such as sulfation, glycosylation, and amidation, among others (116). Further, these properties and amino acid differences between proteins of the same family (117) may stabilize proteins at intermediate states dur- ing thermal stress to prevent irreversible denaturation resulting in proteins that refold partially back to their original states (118). Therefore, a thermal shift assay would be less than ideal to investigate inhibitor–toxin interactions for such thermo- stable proteoforms. However, thermal-based target decon- volution studies are still suitable for a wide range of unique systems including the study of venom metalloproteins in the presence of chelating agents, for example, DMPS and dimercaprol, both of which have demonstrated therapeutic potential towards treating snakebite (49, 115). For example, it was recently demonstrated that TPP with chelating agents can be used to identify novel metal-binding proteins (74). Based on the target family-level thermal properties determined by TPP, we refined our PISA assay parameters to a more sensitive thermal window for target identification and showed that a narrower thermal window selection can improve inhib- itor target identification. These findings highlight how knowl- edge of general thermal properties of a toxin family of interest might be used to improve target identification, perhaps even for protein families with higher thermal stability. between classes. C, heatmap of sum-normalized intensity values in t-treated venom or vehicle control showing concentration-dependent ize variation in sum-normalized intensity between classes. D, correla- n the effects of marimastat treatment on SVMP proteoform intensity. ipitate and supernatant of (E) the most abundant SVMP proteoform centrations of marimastat at the narrow thermal window. SVMP, snake Mol Cell Proteomics (2024) 23(6) 100779 15 Target Specificity of Marimastat Against Snake Venom Toxins Our results demonstrate how analysis of the composition of both supernatants and pellets can be complementary and thus be integrated to further refine inferences of molecular targets (78, 79). In our experiments, we observed varying performances between supernatant and pellet data in the consistent identification of inhibitor targets, particularly of the high abundance SVMP proteoform VAP2B. We found that precipitated material of the narrowed thermal window pro- vided enhanced sensitivity for target deconvolution of the most abundant toxins and across the proteome in general. As previously noted, precipitated material produces better signal- to-noise ratios and more apparent stability ratios than analysis of supernatant (78). Indeed, some previously investigated well-known drug targets were only identified in the precipi- tated material, with no corresponding stability ratio shift in the supernatant (78), as seen with VAP2B in our study. These findings indicate that pelleted material is not just comple- mentary to supernatant-based results but may also be critical for thorough target deconvolution. This is likely due to the continued presence of many proteins even at high tempera- tures as observed in this study and in previous studies (78). We also noted differing performances of the two tested mar- imastat concentrations on target identification, where the higher concentration provided both a higher number of possible SVMP targets and less noise than the lower con- centration of marimastat. In addition to providing information about direct target in- teractions, PISA also allows for off-target effects to be investigated. Off-target binding of a drug may result in adverse effects that decrease (or complicate) its therapeutic utility (114, 119), and small molecule drugs in particular tend to bind a myriad of molecular targets (120). For example, inhibitors of serine proteases exist that may be effective against medically significant SVSPs, but they may also cross-react with endogenous serine proteases in human plasma, which are critical for normal coagulation cascade activation (58). Our PISA analyses identified evidence of the putative interactions between marimastat and off-target toxin families, including CTLs and PLA2 toxins, which were also supported by our liquid chromatography and gel electrophoresis results. These findings are also consistent with prior studies that have shown marimastat and another SVMP inhibitor, prinomastat, can reduce PLA2-based anticoagulant venom effects (54). CTLs have been shown to complex with SVMPs (121), suggesting that there could be a stabilizing effect in conjunction with SVMPs. PLA2s are medically significant enzymes that tend to be fairly ubiquitous and abundant across diverse snake venoms (24, 122–124). Though we did not detect any reduc- tion in PLA2 activity in marimastat-treated samples (data not shown), off-target effects should be considered when inves- tigating small molecule inhibitors of snake venom toxins, as they may demonstrate effects on other medically significant targets and/or contribute to unexpected outcomes in vivo. 16 Mol Cell Proteomics (2024) 23(6) 100779 Multiple snake venom gene families have undergone sub- stantial gene family expansion, diversification, and neo- functionalization that has in many cases resulted in elevated rates of nonsynonymous substitutions in regions of these proteins that determine biological function (12). This trend has been observed in SVMPs (125), SVSPs (126), PLA2s (127, 128), and 3FTXs (129) and has resulted in large multi-gene toxin families with similar structure but a wide array of bio- logical functions and pharmacological effects which can also vary substantially across species (5, 9, 11, 13, 130). Indeed, this diversity of proteoforms within and across species pre- sents an extreme challenge for the development of effective therapeutics to target the effects of these diverse and species- specific toxin cocktails. A major step to addressing this challenge has resulted in efforts to identify the most bioactive and medically relevant toxic proteins and proteoforms in venom using “omics” technologies, which have been referred to as “toxicovenomics” (131–134). A PISA-based approach in combination with toxicovenomics has the potential to take the key next step to address this complex problem through the screening of molecules that may neutralize the action of venom toxins across a wide variety of species that display high variability of medically significant venom toxin families, proteoforms, and activities. PISA and other high-throughput approaches provide promising paths forward for screening of large numbers of commonly studied and currently unex- plored inhibitors against a wide scope of venoms for more rapid development of alternative snakebite therapies. DATA AVAILABILITY The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD046399. Supplemental data—This article contains supplemental data. Acknowledgments—This work was funded in part by the Department of Defense (grant number ID07200010-301-35). Author contributions—C. F. S., T. A. C., Stephen P. Mack- essy, K. C. H., and A. J. S. conceptualization; C. F. S., C. M. M., D. C. G., K. Y. L., Sean P. Maroney, L. B., B. W. P., M. C. M., B. L., and A. J. S. data curation; C. F. S., C. M. M., B. W. P., M. C. M., D. P., B. L., J. J. C., Stephen P. Mackessy, K. C. H., and A. J. S. formal analysis; C. F. S., N. P. B., and A. J. S. funding acquisition; C. F. S., N. P. B., M. C. M., D. P., B. L., T. A. C., Stephen P. Mackessy, K. C. H., and A. J. S. investiga- tion; C. F. S., C. M. M., D. C. G., K. Y. L., Sean P. Maroney, L. B., B. W. P., M. C. M., D. P., B. L., T. A. C., Stephen P. Mackessy, K. C. H., and A. J. S. methodology; C. F. S. and A. J. S. project administration; C. F. S., B. L., J. J. C., T. A. C., Stephen P. Mackessy, and A. J. S. validation; C. F. S., T. A. C., Target Specificity of Marimastat Against Snake Venom Toxins Stephen P. Mackessy, and A. J. S. visualization; C. F. S., C. M. M., B. L., T. A. C., Stephen P. Mackessy, K. C. H., and A. J. S. writing–original draft; C. F. S., C. M. M., B. W. P., M. C. M., D. P., B. L., J. J. C., T. A. C., Stephen P. Mackessy, K. C. H., and A. J. S. writing–review and editing; A. J. S. supervision. Conflict of interest—The authors declare that they have no conflicts of interests with the contents of this article. Abbreviations—The abbreviations used are: BPP, bradyki- nin-potentiating peptide; CRISP, cysteine-rich secretory pro- tein; CTL, C-type lectin; CV, coefficient of variance; FA, formic acid; HPLC, high-performance liquid chromatography; L-AAO, L-amino acid oxidase; LC-MS/MS, liquid chromatography- tandem mass spectrometry; PISA, proteome integral solubil- ity alteration; ProSAP, Protein Stability Analysis Pod; RP- HPLC, reverse-phase high-performance liquid chromatog- raphy; SAR, soluble abundance ratio; SVMP, snake venom metalloprotease; SVSP, snake venom serine protease; TPP, thermal proteome profiling; UNC, University of Northern Col- orado; VEGF, vascular endothelial growth factor. Received November 12, 2023, and in revised form, April 9, 2024 Published, MCPRO Papers in Press, April 27, 2024, https://doi.org/ 10.1016/j.mcpro.2024.100779 REFERENCES 1. Gutiérrez, J. M., Calvete, J. J., Habib, A. G., Harrison, R. A., Williams, D. J., and Warrell, D. A. (2017) Snakebite envenoming. 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Toxicon 138, 119–129 Mol Cell Proteomics (2024) 23(6) 100779 17 http://doi.org/https://doi.org/10.1016/j.mcpro.2024.100779 http://doi.org/https://doi.org/10.1016/j.mcpro.2024.100779 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref1 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref1 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref1 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref1 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref2 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref2 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref2 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref3 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref3 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref3 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref4 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref4 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref4 http://refhub.elsevier.com/S1535-9476(24)00069-0/sref4 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