Biology subjects

Mackessy, S. P.

Publications and source records attributed to Mackessy, S. P..

6 recordsLinked to original sources

bioRxiv2026

De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo

Generative protein design can now rapidly produce de novo binders with high affinity and functional activity against a wide range of targets, including lethal snake venom toxins. However, so far most reported successes rely on new-to-nature scaffolds with limited therapeutic precedent. Single-domain antibodies (VHHs) offer a clinically validated alternative scaffold that can bind and neutralize long-chain -neurotoxins, which are some of the most lethal components in snake venoms. Here we compare three recently established de novo design models with VHH-design capabilities (Germinal, RFantibody, and BoltzGen) for their ability to generate VHHs against the neurotoxin -cobratoxin from the monocled cobra (Naja kaouthia). Using standardized model inputs and evaluation criteria based on AlphaFold3 interface confidence (ipTM) and RMSD self-consistency, we find that Germinal was the only method to generate designs passing stringent in silico criteria for experimental testing. We therefore performed a larger Germinal design campaign employing three different VHH frameworks and experimentally validated 46 designs in vitro. Of these, 42 expressed as soluble proteins and we identified four binding hits derived from two of the three tested frameworks. Of the four binders, two lead candidates were further characterized and demonstrated high affinity (KDs of 4.1 nM and 10.8 nM), monomeric behavior and low polyreactivity, indicating favorable biophysical and developability properties, as well as functional toxin neutralization in vitro. To assess their therapeutic potential we investigated their ability to protect against -cobratoxin toxicity in vivo. Both candidates fully protected mice after -cobratoxin challenge, with 100% survival compared to a lethal control. One candidate also retained notable neutralization capacity against whole venom of Naja kaouthia with a survival of 56%, while the other protected 22% when tested in a rescue setting. Together, we demonstrate that de novo VHH design can generate high affinity single-domain antibodies with in vivo protection against lethal cobra venom neurotoxicity, and provide practical insights into method- and framework-dependent performance.

bioengineering↗

bioRxiv2026

Establishment of snake venom gland organoids from a novel family, Colubridae

Non-front-fanged snakes are abundant, diverse and represent approximately 70% of extant snakes. However, there is limited knowledge about most species and their venoms, in part due to the technical and welfare challenges associated with venom extraction, low venom yields, and the lack of cellular models available. Organoids represent an excellent opportunity to overcome these challenges. Here, we establish, for the first time, venom gland organoids from snakes of the Colubridae family and demonstrate the in vitro production of toxins.

bioengineering↗

bioRxiv2024

Discerning Specific Thrombolytic Activities and Blood Clot Degradomes of Diverse Snake Venoms with Untargeted Peptidomics

Identification and characterization of snake venom toxins that interfere with hemostasis have important implications for the treatment of snake envenomation, the bioprospecting of therapeutically useful molecules, and the development of research tools for investigating hematologic disorders. Many venoms have been shown to possess thrombolytic activity. However, it remains unclear if actions on other clot-stabilizing proteins beyond fibrin chains contribute significantly to venom-induced thrombolysis because the clot-wide targets of venom proteases and the mechanisms responsible for thrombolysis are not well understood. Here, we utilize a high-throughput time-based thrombolysis assay in combination with untargeted peptidomics to provide comprehensive insight into the effects of venom from six snake species on blood clot degradation. We compare thrombolytic profiles across venoms with variable levels of proteases and generate venom-specific fingerprints of cleavage specificity. We also compare the specific effects of venoms that possess a range of thrombolytic activity on fibrin subunits and other clot-bound proteins involved in clot structure. Venoms with higher thrombolytic activity demonstrated an enhanced ability to target multiple sites across fibrin chains critical to clot stability and structure, as well as clot-stabilizing proteins including fibronectin and vitronectin. Collectively, this study significantly expands our understanding of the thrombolytic and fibrinolytic effects of snake venom by determining the full suite of clot-specific venom targets that are involved in clot formation and stability.

pharmacology and toxicology↗

bioRxiv2023

ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization 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 outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including Thermal Proteome Profiling (TPP) 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 TPP 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 characterize 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 thermal window and soluble supernatant with insoluble precipitate 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.

biochemistry↗

bioRxiv2023

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes

Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulation in elapids is largely unexplored. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans- acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). Although there is conservation in high-level mechanistic pathways regulating venom production, there are histone methylation, transcription factor, and microRNA regulatory differences between these two snake families. Histone methyltransferases (KMT2A, KMT2C and KMT2D) and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target different toxin transcripts. Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin evolution between these two snake families, demonstrating multiple toxin genes and regulatory mechanisms converged to underpin a highly venomous phenotype.

evolutionary biology↗

bioRxiv2022

Relictual Hybridization and Biogeography of Massasauga Rattlesnakes (Sistrurus spp.)

Climate change inevitably leaves behind a genetic footprint within phylogeographic legacies of affected species, as individuals are driven to either disperse to track suitable conditions or adapt in situ. One potential consequence is the possibility of hybridization among species, as both geographic ranges and adaptive landscapes shift. The admixture resulting from these newly formed contact zones has various outcomes, to include the creation of new lineages. Interpreting these within the context of historic climate change provides clues necessary to predict biotic responses (and thus evolutionary trajectories) as a function of contemporary shifts. Herein, we dissect historic contact zones for Massasaugas (Viperidae; Sistrurus spp.) within two distinct North American regions (southwestern United States and Central Great Plains) using ddRAD sequencing. We identified fine-scale but previously unrecognized population structure within the southwestern contact zone, where we detected contemporary intergradation between Prairie and Desert massasaugas (S. tergeminus tergeminus, and S. t. edwardsii, respectively), with primary divergence indicated by demographic model selection. Within the Central Great Plains, we found evidence for historic secondary contact via Quaternary climatic cycles, subsequently followed by range expansion at the suture zone separating S. tergeminus and S. catenatus. Extant Missouri populations represent ancestral/relictual vestiges of this earlier hybridization, isolated between the eastern terminus of S. t. tergeminus and the western edge of S. catenatus. Our results illustrate how abrupt climate change has driven ancestral hybridization, cryptic diversity, and range dynamism within Sistrurus.

evolutionary biology↗