INTRODUCTION

The modern therapeutic toolbox has been dominated by two poles. Small molecules excel at oral dosing, distribution, and manufacturing, and they are superb when a target exposes deep, well defined pockets. Yet their very compactness, typically <600 Daltons, constrains the surface area they can contact, making it difficult to modulate large, relatively flat protein–protein interfaces (PPIs) or to discriminate among closely related binding epitopes.

At the other pole, biologics (antibodies, proteins, and related modalities) bring tremendous shape complementarity and affinity to accessible extracellular targets. But macromolecular size, charge, and trafficking obstacles limit efficient penetration into cells, which is why biologics have historically focused on extra­cellular or lumenal proteins; cytosolic targets remain challenging without specialized delivery tricks.

Small molecules remain the workhorses of medicine, from statins, painkillers, antibiotics, to kinase inhibitors, but they rely on highly complementary pockets and well behaved absorption, distribution, metabolism, and excretion. When the thermody­namic currency of binding is spread over hundreds of square angstroms, as in many PPIs, small molecules often cannot make enough contacts without becoming too bulky or polar to be “drug like”. Biologics, by contrast, bring tremendous shape complemen­tarity and affinity to accessible extracellular epitopes, but struggle to reach intracellular targets at pharmacologically useful concen­trations.

Peptides occupy the space in between. They inherit the se­quence programmability of proteins, yet can be engineered to approach the permeability and stability of small molecules. They present more binding surface than small molecules and, unlike large proteins, can sometimes be tuned to cross membranes, oc­cupy shallow clefts, or clamp onto protein surfaces. The key chal­lenge, and opportunity, is to sculpt peptide architecture so that shape and chemistry are presented to the target in the right place, at the right time, and for long enough to matter clinically.

 STRUCTURE EQUALS FUNCTION: FROM BIOLOGY 101 TO DRUG DESIGN

One of the first durable lessons in biology is that structure determines function. Proteins fold; the fold creates pockets, sur­faces, and defined electrostatics; those features control catalysis and recognition. Drug design is no different: a therapeutic’s topology, the way its atoms are organized in space, often dictates its fate. Yet most peptide discovery platforms today search li­braries that explore sequence far more than architecture. Many screens limit candidates to a single macrocycle or a narrow set of constrained motifs, which restricts the exploration of shape space.

Consider insulin – the clinically used hormone comprises two chains (A and B) held by disulfide bonds. Efforts to collapse insulin into a single chain receptor agonist underscore how hard it can be to recapitulate a multi segment architecture with fewer con­nections. The broader point is that architecture is causal and lim­iting architecture up front can limit what biology you can access. However, there are two root cause problems in peptide therapeu­tics:

Problem 1: Searching Astronomical Sequence Space : High throughput selection technolo­gies have transformed discovery. mRNA display covalently links a nascent peptide to its mRNA via a puromycin “bridge,” en­abling in vitro selections over libraries that can exceed 1012 distinct sequences. Phage display genotypically couples peptides to bacteriophage coat proteins (commonly pIII or pVIII, two of the viral “appendages”) for iterative biopanning. DNA encoded li­braries (DELs) extend the barcode concept to small molecules and peptidomimetic search spaces. Each platform makes it possible to sift through large diversity be­cause the barcode (DNA/RNA) can be am­plified from an incredibly small signal.

In each display system, the barcode is the engine: it ties genotype to phenotype and allows exponential amplification of faint signals by PCR or phage replication. That same barcode, however, can perturb physics at the interface. In mRNA display and DELs, long, polyanionic nucleic acids can electrostatically repel negatively charged patches on targets or nonspecifi­cally attract basic surfaces. In phage dis­play, the per virion copy number, orientation, and spatial crowding on the capsid can alter the apparent fitness of certain peptide topologies versus their be­havior as free molecules.

Problem 2: Conformational Control (the “Floppiness” Problem): Linear peptides are intrinsically disor­dered. Without constraints, they can adopt many conformations, only a small fraction of which align with the bioactive pose, lowering apparent affinity and making them susceptible to proteolysis. Chemistry has long fought back with macrocycliza­tion, historically via disulfide bridges, then with lactams, ring closing metathesis, click reactions, and staples. These strategies often stabilize a single ring. They improve stability and sometimes permeability, but they inherently limit architectural diversity because only a few bond forming reac­tions are both reliable and library compat­ible across many sequence contexts.

NATURE’S BLUEPRINT: ENZYMES THAT BUILD RIGID, BIOACTIVE SCAFFOLDS

Nature points to a practical way for­ward. Ribosomally synthesized and post translationally modified peptides (RiPPs) are encoded as linear precursors and then sculpted by tailoring enzymes into com­pact, diverse scaffolds with exceptional stability and activity. Among the most cel­ebrated are lantibiotics, such as nisin, which uses multiple thioether (sulfur-to-carbon) bridges to achieve food grade sta­bility and long-term potency. Beyond lanthipeptides, the radical S adenosyl L methionine (rSAM) superfamily forges many unique radical-based transforma­tions in diverse contexts, including gener­ating thioethers in peptide contexts.

Two families of thioether rich RiPPs illustrate how chemistry grants stability. Lan­thipeptides (eg, nisin) are built by first de­hydrating serine and threonine residues to dehydroalanine/dehydrobutyrine and then adding generate the thioether through a Michael-addition via a cysteine residue to form lanthionine/methyllanthionine bridges; bonds that resist reduction where disulfides would not. Sactipeptides/ranthipeptides use rSAM en­zymes to forge C-S bonds directly to aliphatic carbons, again delivering robust thioether linkages.

A standout example is PapB, an rSAM RiPP maturase that installs thioether cross links between Cys and Asp/Glu residues. In its native substrate PapA, PapB intro­duces six thioether linkages, and in-vitro studies show striking substrate promiscuity: PapB accepts a range of Cys Xₙ Asp (or Cys Xn Glu, were Xn is a number of addi­tional amino acids) motifs, can form nested and in line cross links, and tolerates unnatural amino acids at the cross linking positions. These features suggest that en­zyme installed linkages could decouple se­quence from topology, enabling many ring architectures beyond what chemical meth­ods comfortably deliver.

There is a catch: most rSAM enzymes are oxygen sensitive because their [4Fe 4S] clusters are labile. Practical deployment at discovery scale therefore requires anaero­bic handling and careful attention to re­ducing systems during expression, purification, and catalysis.

WHAT IT TAKES TO EXPLOIT ENZYMATIC MACROCYCLIZATION AT HTS SCALE

To translate these natural capabilities into a discovery engine, three require­ments must be met: (1) Enzyme tolerance to library diversity, so that billions of unique sequences with cross link motifs can be processed, (2) Evidence of cycliza­tion in HTS contexts, demonstrating that cross links can be installed on constructs used for phage and mRNA display, and (3) Fast topology confirmation, so that hits can be prioritized by architecture as well as se­quence.

For topology confirmation, mass spectrometry can diagnose the character­istic 2 Da mass loss per thioether, judicious isotopic labeling, and protease mapping can rapidly fingerprint ring count and con­nectivity. On target functional triage should begin early (eg, receptor activation assays) because architectures that bind but do not signal are often unhelpful for ther­apeutics. Combining these readouts with next generation sequencing (NGS) enables structure aware enrichment curves in which rises and falls in specific ring pat­terns are tracked alongside sequence fre­quency.

SETHERA’S APPROACH: DECOUPLING TOPOLOGY FROM SEQUENCE

Sethera Therapeutics, Inc. is develop­ing an enzymatic platform that decouples topology from sequence, enabling control­lable, site specific multi ring formation under biological conditions while preserv­ing chemical handles for downstream con­jugation. The working model is straightforward: encode CXₙD/E motifs within diverse peptide backbones, allow PapB like rSAM enzymes to install thioether cross links, and carry out selections on the cyclized, barcoded products.

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Early cell free biosynthesis (CFB) ex­periments and pIII based phage selections already hint at architecture specific enrich­ment, consistent with the notion that topol­ogy, nested versus in line versus triple, can dominate fitness during selection. The platform keeps orthogonal chemical han­dles available (eg, N termini, C-termini or tailored side chains) so that post selection conjugations (fluorophores, affinity tags, payloads) can be attached without disturb­ing the enzyme set cross links. Together, this enables rapid hit confirmation and assay portability across formats.

To reduce format bias, discovery is run in two orthogonal display contexts on the same targets. mRNA display provides ultra large sequence spaces with a polyan­ionic barcode (RNA/DNA) that can, in some cases, shift electrostatics near the binding interface. Phage display imposes capsid sterics (eg, pIII spacing, avidity, and orientation) that can mask or exaggerate certain architectures. By selecting an mRNA display and then reformatting en­riched pools into phage for orthogonal/parallel screens, Sethera max­imizes search breadth while minimizing format artifacts.

Why explicitly combine both HTS sys­tems? Because each has systematic arti­facts. In mRNA display, the long nucleic acid barcode can create electrostatic halos that disfavor some topologies or select for peptides that bind in geometries compati­ble with the tag. In phage, virion proximity effects and copy number can favor archi­tectures that survive secretion and display constraints but are not necessarily optimal in free solution. Convergent enrichment across both formats flags architectures that are more likely to be intrinsically fit, and thus, more likely to survive translation into therapeutic scaffolds.

THE WAY FORWARD: FROM BINDERS TO FUNCTIONAL MODULATORS

Polymacrocyclic peptides are more than “better binders”. Multiple rings can pre organize pharmacophores, enforce multivalent effects within a single chain, and bias conformational dynamics in ways that translate into signaling outcomes; for example, agonism versus antagonism at GPCRs or isoform specific engagement of homologous domains. Sethera’s develop­ment path emphasizes:

Library Design by Topology: construct families that systematically vary ring count, spacing, and order (nested, in line, and triple ring motifs), not just side chain iden­tities.

Orthogonal Discovery: perform parallel mRNA and phage selections on identical targets to identify architectures that repro­duce in both contexts.

Rapid Structure Confirmation: use MS/MS fingerprints and enzymatic diges­tion to verify cross link count and place­ment early, accelerating hit triage.

Functional Assays, Not Only Binding: prioritize cell based readouts (activation, inhibition, internalization, trafficking) to learn which architectures modulate biol­ogy, not merely associate with targets.

Developability Filters: bake in plasma/microsomal stability and perme­ability screens; leverage the inherent re­duction resistance of thioether bonds relative to disulfides, and preserve orthog­onal chemical handles to enable payload conjugation or depot strategies.

Polymacrocyclic peptides can address intracellular protein-protein interactions long considered “undruggable” to small molecules and too hidden for antibodies; they can be tuned to bias receptor signal­ing or recruit endogenous machinery (eg, for targeted degradation or trafficking). Most importantly, they let us search the middle ground by structure, where the best medicines for complex diseases may be hiding.

The field has long known that shape underlies function, but our discovery en­gines have searched sequences far more than shapes. Enzyme installed polymacro­cyclization changes that calculus. By merg­ing RiPP enzymology with genetically barcoded discovery and orthogonal selec­tion formats, we can systematically explore topology as a drug variable. Sethera’s platform is designed to do exactly that, turning nature’s blueprint into a practical path to specific, stable, and effective pep­tide therapeutics.

Dr. Karsten Eastman is the CEO of Sethera Therapeutics, Inc. He earned his PhD from the University of Utah (under the mentorship of Professor Vahe Bandarian), where they discovered the key processes that enable Sethera Therapeutics’ innovative technology. As a co-founder of Sethera, Dr. Eastman is leading the company’s efforts to commercialize cutting-edge peptide therapeutics, drawing on their experience in enzyme and peptide research, patent development, and engagement with key business mentors.

 

Dr. Vahe Bandarian is the CSO and President of Sethera Therapeutics, Inc. He earned his PhD in Biochemistry from the University of Wisconsin-Madison. He is a distinguished faculty member and Associate Dean at the University of Utah, recognized for his exceptional contributions to the field of chemistry. He is a Fellow of the American Chemical Society (ACS), the American Association for the Advancement of Science (AAAS), and the American Society for Biochemistry and Molecular Biology (ASBMB). His groundbreaking research has earned him the prestigious Pfizer Award in Enzyme Chemistry from the ACS and the Distinguished Research Award from the University of Utah.