Treating Fungal infections with synthetic peptide mimics

Modern medicine often relies on invasive medical interventions or drugs that can compromise the patient’s immune system. An unfortunate consequence of these undeniably successful treatments for life-threatening diseases like cancer is the occurrence of severe infections caused by opportunistic fungal pathogens, including species of Candida, Aspergillus, Cryptococcus, and Pneumocystis. More recently, increasing numbers of opportunistic fungal infections caused by Aspergillus, Mucorales, and Candida species have been observed in COVID-19 patients with severe respiratory syndromes in intensive care units. These factors, among others, have resulted in over 2 million invasive fungal infections annually worldwide, with alarmingly high mortality rates, causing more than 1.5 million deaths.

Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. In a recent publication by Schaefer, S., Vij, R., Sprague, J.L. et al., they highlight the enormous potential of synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.

Schaefer, S., Vij, R., Sprague, J.L. et al. A synthetic peptide mimic kills Candida albicans and synergistically prevents infection. Nat Commun 15, 6818 (2024). Link to publication

Trishul Shah
Global Director Sales and Marketing, PolyPeptide Group
Member, BPF Scientific Advisory Board
www.linkedin.com/in/trishul-shah-bb92a2/

Read previous editions of the BPF Journal Club series: https://www.boulderpeptide.org/journal-club

A Knotty Challenge:  Novel cystine-knot peptide inhibitors of HTRA1

The July BPS journal club features an article showcasing the power of cystine-knot peptides (CKPs) as high-affinity and high-specificity protein inhibitors. A team from the laboratories of Rami Hannoush and Daniel Kirchhofer at Genentech created diversity libraries based on the carboxypeptidase A1 inhibitor and Ecballium elaterium trypsin inhibitor II CKPs. They performed phage display and affinity maturation studies to select potent inhibitors of a different serine protease, HTRA1 (https://doi.org/10.1038/s41467-024-48655-w)¹. The authors then characterized their modes of binding in structural studies and exploited differences in this binding pocket between serine protease family members to increase selectivity to the target. Specificity is an especially challenging problem in the serine protease family due to the large number of related family members with a fairly conserved binding pocket, including four highly related members of the HTRA family, HTRA1-4.

HTRA1, or high-temperature requirement A serine peptidase 1, is a trimeric serine protease implicated in several disorders, including arthritis, osteoporosis, age-related macular degeneration, Alzheimer’s, and chemoresistance. Both antibody-based (https://doi.org/10.1073/pnas.1917608117)² and small molecule, peptidomimetic-like inhibitors of HTRA1 (https://doi.org/10.1016/j.bmcl.2024.129814)³ have been identified, and clinical studies have been initiated with a Fab inhibitor of HTRA1 for age-related macular degeneration (NCT03972709).

Cystine-knots are structural motifs in proteins comprising three disulfide bridges between cysteine amino acids, where one strand or loop passes through another loop, forming a rotaxane structure that is protease resistant and shows both chemical and thermal stability. The core portions of CKPs have relatively low molecular weights (generally around 30 amino acids and less than 4 kDa) and are naturally occurring in animals, plants, and insects. Some common examples include nerve growth factor, transforming growth factor-beta, and various venom peptides from snails, spiders, and scorpions. They can also be chemically synthesized or manufactured using biological expression systems, making them excellent candidates for therapeutics.

The authors generated phage peptide libraries by introducing amino acid randomization and length variation into the surface-exposed loops of the CKP scaffolds and screened the resulting phage for binding to HTRA1 constructs by phage ELISA. Hits from the initial screen were modified through affinity maturation studies, and several inhibitors were identified with single-digit nanomolar affinities that also showed IC₅₀s for HTRA1 activity on a substrate in the same range. Biochemical studies were then used to map the approximate binding regions for the compounds on HTRA1, followed by structural studies that demonstrated that the CKP inhibitors bind a cryptic pocket near the active site and stabilize a structure that renders HTRA1 non-competent for protease activity. This cryptic pocket showed some differences from other members of the HTRA protease family, and the authors used a combination of structure-based design and further affinity maturation to exploit these differences and generate inhibitors with both high potency and high selectivity for HTRA1, even when compared to HTRA2, 3, and 4.

This article is a fantastic example of collaborative, multidisciplinary work using genetics, biochemistry, and structural biology to identify peptide inhibitors with high affinity and selectivity for a challenging target. Furthermore, it is another example of optimizing naturally occurring structures to develop candidate therapeutics that have the potential to be stable and well-tolerated since there are numerous examples of CKPs in biology. I look forward to seeing the effects of these inhibitors in animal models of diseases related to HTRA1 in the near future, as well as publications using a similar approach to identify cystine-knot peptide inhibitors of other challenging targets.

Dave Garman, PhD
Member, BPF Scientific Advisory Board

¹Li, Yanjie et al.  Cystine-Knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.  Nature Communications 15:4359 (2024)

²Tom, Irene et al.  Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.  PNAS 117(18):9952-63 (2020)

³Dennis, David G et al.  Identification of highly potent and selective HTRA1 inhibitors.  Bioorganic & Medicinal Chemistry Letters 109, September 2024 (available online ahead of print)

Read previous editions of the BPF Journal Club series: https://www.boulderpeptide.org/journal-club

Green Chemistry in Peptide Therapeutics: Leading the Way from the Beginning

In recent years, the field of peptide therapeutics has seen a surge of interest in cyclic peptides, driven by their unique properties and potential as active pharmaceutical ingredients (APIs). Cyclic peptides are particularly appealing due to their ability to be designed with desirable membrane solubility and oral absorption properties, which opens up the opportunity to target intracellular receptors and proteins effectively.

One of the key advantages of cyclic peptides is their rigid structure, which presents a large surface area capable of interacting with protein-protein interfaces. This makes them ideal candidates for disrupting protein interactions that are otherwise challenging to target with small molecules.

The development and screening of complex cyclic peptide libraries, especially those incorporating non-canonical amino acids, have become standard practices in many research laboratories. This advancement is exemplified by companies like Peptidream and RA Pharma, which have demonstrated success using mRNA libraries. The incorporation of non-canonical amino acids significantly expands the diversity and properties of these peptides, overcoming the limitations of the 20 natural amino acids.

However, once a promising lead is identified, scaling up the production of these peptides presents significant challenges. The synthesis of large quantities of non-canonical amino acids required for clinical programs is often complex and expensive, potentially delaying projects and increasing costs. To address this, several major pharmaceutical companies are investing in innovative platforms for the production of non-canonical building blocks that are both scalable and cost-effective.

An excellent example of such innovation is highlighted in an open-access article by Merck & Co. Inc. The article outlines a general approach for producing beta-branched aryl chiral amino acids using biocatalysis. Merck’s approach focuses on three key prerequisites: i) conducting reactions in water, ii) using an enzyme with broad selectivity and high chiral specificity, and iii) utilizing a cheap and readily available substrate as the amino donor.

In their study, Merck successfully used lysine as an amino donor, racemic aryl keto acids as the beta-branched starting materials, and transaminase as the enzyme to achieve high-yield, stereoselective reactions. Remarkably, the reactions proceeded with yields averaging above 70%, consistently producing the S,S configuration.

These beta-branched amino acids are now likely being incorporated into cyclic peptide libraries produced by Merck and its partners. This development is particularly exciting because the ability to lock the conformation of sidechain rotamers is a critical parameter for the success of these libraries.

In our April Journal Club edition, we discussed the necessity for peptide chemistry to become lean and green, emphasizing the adoption of green chemistry early in the API lifecycle. Merck’s efforts exemplify this principle by integrating green chemistry into their lead compound library screening process (we hope), ensuring that the compounds selected can be produced sustainably.

This article from Merck provides a compelling case study of how green chemistry can be implemented effectively in peptide therapeutics, paving the way for more sustainable and efficient drug development processes.

Article Citation: Dunham N, Ray R, Eberle C, Winston M, Newman J, Gao Q, et al. Efficient Access to β-Branched Noncanonical Amino Acids via Transaminase-Catalyzed Dynamic Kinetic Resolutions Driven by a Lysine Amine Donor. ChemRxiv. 2024; doi:10.26434/chemrxiv-2024-qm6pl. This content is a preprint and has not been peer-reviewed. The authors have deposited a ChemRxiv open-access version at https://chemrxiv.org/engage/chemrxiv/article-details/660da6f221291e5d1dfe8287.

Matteo Villain, PharmD.
Member, BPF Scientific Advisory Board
linkedin.com/in/matteo-villain

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This Pub. has the Guinness…for longest synthetic peptide!

In the May installment of the BPS Journal Club, I’d like to highlight a recent paper from the Walczak group (in Boulder!) that addresses a beast of a target, a microtubule-associated protein: Tau.

A 441-amino acid tour de force of chemical synthesis! There are plenty of nuggets here for chemists and biologists alike. If you're looking to assemble large peptides, or are in search of point mutations without the limitations of post-translational tools using the native sequences, or just simply curious of the odyssey, this publication will be a treat.

Tau is a focus point in studying neurodegenerative diseases, as they are characterized by intraneuronal deposits of this protein, hence their collective name, tauopathies. Tau exists in several variants and is prone to a vast array of post-translational modifications (PTMs). Examining the role of PTMs in tauopathies is thus a hot topic but is a challenging one in the
absence of a fully synthetic route to the protein. Semi-syntheses and synthetic PTMs of the native variants are limited to specific sites and substitutions.

The authors here propose a route to the longest isoform of the six Tau, by fragment-based chemical assembly. A retrosynthetic analysis identified three key fragments of ca. 150 amino acids each, which were further dissected into subunits <50 AA in length, at strategic cysteines or alanines. The preparation of each fragment then unfolds as a who's who of modern
SPPS techniques to obtain the desired fragments: resin/linker selection, protecting group selection, hydrazide approaches to thioesters or their surrogates, all while keeping the more conventional stowaways, such as aspartimides, at bay. Some fragments took a few tries. Some did not cooperate at the ligation stage and required rework. But eventually, the team had all the ingredients for the final native chemical ligations, both with sulfur and selenium, and the adequate sprinkle of desulfurizations.

This monumental synthesis is a strong one-up on the already formidable GFP precursor synthesis (Sakakibara et al, 238 AAs), not that there should be a competition! It should pave the way for more accessible, on-demand PTMs of long peptides and small proteins for the benefit of biologists and chemists alike.

The paper is available at JACS: Chemical Synthesis of Microtubule-Associated Protein Tau (https://pubs.acs.org/doi/10.1021/jacs.3c07338).

The authors have deposited a ChemRxiv open-access version as well (https://chemrxiv.org/engage/chemrxiv/article-details/64a7639f9ea64cc167918c5d).

Alaric Desmarchelier, Ph.D.
Member, BPF Scientific Advisory Board
https://www.linkedin.com/in/alaric-desmarchelier/

Read previous editions of the BPF Journal Club series: www.boulderpeptide.org/journal-club

Can Solid Phase Peptide Synthesis Learn to Become Leaner?

For the April edition of the BPF Journal Club, I would like to introduce an extremely interesting communication by Collins and co-workers from CEM Corporation (https://doi.org/10.1038/s41467-023-44074-5). The subject is of particular interest to those involved in peptide chemistry, peptide drug discovery, and manufacturing. The work addresses important issues related to solid-phase peptide chemistry (SPPS), in the context of large-scale production.

Since its introduction in the early 1960’s SPPS has revolutionized peptide science, especially the discovery and development of peptide therapeutics. Recent years have witnessed the emergence of incretin-based diabetes/obesity therapeutics which, as a result of unprecedented demand, require large-scale manufacturing. While some, including liraglutide and semaglutide (both from Novo-Nordisk), can be made in part by recombinant expression, others, notably tirzepatide (Eli Lilly & Co.), rely entirely on chemical synthesis. Utilizing SPPS at scales of hundreds of kilograms or more highlights several inherent limitations of the SPPS method. One of these is the poor atom-efficiency resulting from the several-fold stoichiometric excess of Fmoc-protected amino acid and coupling reagent used in each step. The second source of inefficiency involves the high consumption of solvents such as N,N-dimethylformamide (DMF) or N-methyl-pyrrolidone (NMP) used in wash steps between couplings. This produces a large volume of waste and substantially increases disposal costs. Both factors contribute to an unfavorably high process mass intensity (PMI) score (https://doi.org/10.1021/acs.joc.3c01494). This concern is further exacerbated by a regulatory climate that has restricted the use of DMF in the European Union (https://doi.org/10.1002/cssc.202301639).

The authors present an effective solution to the above problem by introducing a novel SPPS protocol that utilizes microwave-assisted evaporation and nitrogen flushing of pyrrolidine following the Fmoc deprotection step. Pyrrolidine is used in place of piperidine due to the former’s lower boiling point (bp 87 degC for pyrrolidine vs. bp 106 degC for piperidine). This modification results in a reduction of solvent and base waste of around 80%. The method is optimized using the Jung Reddman sequence and validated for several peptides including liraglutide and semaglutide, as well as the proteins proinsulin and barstar. The possibility of epimerization was checked by comparing a liraglutide sample made by the new method with a commercial sample noting no significant differences. The new methodology holds promise for minimizing not only production costs but also environmental and regulatory concerns for future large-scale peptide projects.

John P. Mayer, Ph.D.
Member, BPF Scientific Advisory Board
linkedin.com/in/john-mayer-4587556