Protein-Metabolite Interactome Is Vast Untapped Frontier For Drug Discovery

By Deborah Borfitz 

April 13, 2023 | Researchers at the University of Utah have created a systematic approach to reveal the endogenous protein-metabolite interactome at high-throughput speed. The platform quickly identifies metabolites that putatively interact with target proteins, according to Kevin Hicks, Ph.D., a biochemistry research instructor and consultant to Boston-based Atavistik Bio. 

Hicks is co-developer of MIDAS—mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically—along with biochemistry professor Jared Rutter, Ph.D., cofounder of Atavistik. The University of Utah licensed the technology to the biotechnology company, and it serves as the foundation of the Atavistik Metabolite Protein Screening (AMPS) platform informing small molecule drug discovery.  

In a study, published recently in Science (DOI: 10.1126/science.abm34), Hicks and his colleagues used MIDAS to analyze 33 enzymes from human carbohydrate metabolism to identify 830 protein-metabolite interactions. Many of these intrapathway interactions were already known, but many were novel, says Hicks. Given that there are thousands of proteins in the cell, the full scale of the metabolic network is predicted to be much larger. 

The paper highlighted the regulation of lactate dehydrogenase (LDH), an enzyme associated with carbohydrate metabolism that has two isoforms—LDHB, more highly expressed in the heart, and LDHA, more highly expressed in the liver. Interestingly, the research team found that both adenosine triphosphate (ATP) and long-chain acyl-coenzyme A inhibited only LDHA even though the proteins are about 75% identical, says Hicks. 

Follow-up investigations now underway are looking at what this means within different tissues and on the organismal level, he adds. LDHA has apparently evolved to be sensitive to ATP, a nucleotide that is a source of energy for use and storage at the cellular level. “It is known that tissues make decisions to burn carbohydrates or fats, and to do this metabolism is rewired. Inhibition of LDHA by fatty acid intermediates may be one mechanism to facilitate that rewiring.” 

‘Re-envisioned’ Technology 

MIDAS has helped uncover a biological network inside cells that adjusts in real time to withstand various stresses, such as eating a lot of carbs, working out at the gym, or recovering from an illness. It is, as Rutter puts it, “how nature has evolved to ‘drug’ its own proteins and pathways.” 

Analyzing the protein-metabolite interactome could provide clues for making better medicines for metabolic diseases and cancer, says Hicks. It has been an under-studied field relative to other domains of biology, including proteomics and genomics, where the enabling technologies are well established.   

As Hicks describes it, MIDAS is based on a classic biochemistry technique, equilibrium dialysis, which is used to study receptor-ligand interactions but in a comparatively minimalistic way. The technology has been “re-envisioned” to be higher throughput and enable the systematic discovery of protein-metabolite interactions. 

A target protein of interest is confined to a chamber in a 96-well plate and separated by a semi-permeable membrane from a pool of metabolites that can freely diffuse across both chambers, he explains. If any of those metabolites interacts with the target protein, their signal will either go up (enriched) or down (depleted) in the protein chamber relative to the free concentration of metabolites, depending on the nature of the interaction. The abundance of all metabolites gets quantified simultaneously using a high-throughput flow injection analysis mass spectrometry platform traditionally used for metabolomic profiling.  

The approach can be used to assay a target protein, or multiples of them, against hundreds of metabolites simultaneously, continues Hicks. “In relatively short order... it gives you an idea of what endogenous metabolites will interact with that protein.” The next step would be to figure out their role in the cell, such as to changing the protein’s function or its localization. 

New Directions 

Rutter imagined equilibrium dialysis being used for studying the protein-metabolite interactome before Hicks joined the effort in 2016. The lab was piloting low-throughput technology employing a small metabolite library. Recognizing the untapped discovery potential of the technique, Hicks says, is what prompted him to take the technology to the next level with a larger library reflective of the human metabolome. 

Atavistik was founded in 2021 by Rutter and Ralph DeBerardinis, M.D., Ph.D., chief of pediatric genetics and metabolism at the University of Texas Southwestern Medical Center (UTSW) and director of the genetic and metabolic disease program at the Children’s Medical Center Research Institute at UTSW. Its overriding mission is to identify protein-metabolite interactions that can be leveraged as therapeutics for treating inborn errors of metabolism.  

The latest published study, where Rutter is the corresponding author, finds that enzymes from carbohydrate metabolism monitor their metabolic environment and adapt their activity as needed. The plan is to eventually scale the MIDAS technology such that it can screen across the whole proteome, says Hicks, but the initial focus was on the “hub” of metabolism that includes the pathways of glycolysis, gluconeogenesis, and tricarboxylic acid cycle together with some branching pathways. 

“It was a very logical place to start to benchmark this technology because these pathways have been studied for 100-plus years... [and] we know multiple metabolite regulators of some of these enzymes,” as discovered using low-throughput approaches, says Hicks. The study team either collaborated or prepared all of the enzymes and their isoforms and then screened them using the MIDAS platform. 

The investigation revealed a massive interaction network composed of proteins and metabolites arranged on metabolic pathways “like beads on a chain... [with] a bunch of interactions [happening] both upstream and downstream of those enzymes,” he says. A subset of those interactions was functionally validated to show novel regulation as well as “a huge number of interactions from metabolites in metabolically distant pathways... potentially suggesting long-range regulation of this central [carbohydrate] pathway.” 

The technology holds vast potential for revealing functional interactions and how the metabolic state of cells responds acutely at the protein level, says Hicks, who is soon to go on the tenure track position search. MIDAS has been targeting soluble proteins up to now, but he will be developing a new related technology to start looking at membrane protein-metabolite interactions. Membrane proteins make up a significant and unexploited fraction of the proteome, he notes, and are also the target of many drugs approved by the U.S. Food and Drug Administration.