Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis.

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps. Therefore, the Peptide ADME Working Group sponsored by the Translational and ADME Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) was formed with the goal to develop a white paper focusing on metabolism and excretion studies to support discovery and development of TPeps. In this paper, the key learnings from an IQ industry survey and U.S. Food and Drug Administration/European Medicines Agency submission documents of TPeps approved between 2011 and 2022 are outlined in detail. In addition, a comprehensive assessment of in vitro and in vivo metabolism and excretion studies, mitigation strategies for TPep metabolism, analytical tools to conduct studies, regulatory status, and Metabolites in Safety Testing considerations are provided. Finally, an industry recommendation on conducting metabolism and excretion studies is proposed for regulatory filing of TPeps. SIGNIFICANCE STATEMENT: This white paper presents current industry practices for metabolism and excretion studies of therapeutic peptides based on an industry survey, regulatory submission documents, and expert opinions from the participants in the Peptide Absorption, Distribution, Metabolism, and Excretion Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development. The group also provides recommendations on the Metabolites in Safety Testing considerations and metabolism and excretion studies for regulatory filing of therapeutic peptides.

Liposomal Permeation Assay for Droplet-Scale Pharmacokinetic Screening.

Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (Z' ≥ 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and -impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

A solid-phase approach for the synthesis of muramyl dipeptide conjugates for detection of NOD2.

Proper recognition of invading pathogens and prompt initiation of host defense mechanisms are instrumental for the maintenance of organismal homeostasis. Nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs) serve as pathogen-recognition receptors that specifically recognize bacterial peptidoglycans. NOD2 detects muramyl dipeptide (MDP) through its carboxy-terminal leucine rich repeats (LRRs), which enables the activation of downstream inflammatory signaling. Synthesis of MDP conjugates based on solution phase chemistry have been previously reported. Our solid phase approach synthetically provides a facile approach for the conjugation of biological probes to MDP, with the advantage of minimal functional/protecting group manipulation, and reduction in the laborious process of intermediate purification and isolation. MDP conjugates that we generated using solid phase synthesis allow detection of NOD2 is cell lysates and NOD2 subcellular localization by immunofluorescence microscopy. MDP-PEG6-Cyanine5.5 conjugate selectively colocalized with WT NOD2 but not NOD2 variant found in Crohn's disease, which lacks carboxy-terminal end and cannot bind MDP. Overall, these data indicate that distinct solid phase-produced MDP conjugates can be used to examine biological properties of NOD2 and could potentially facilitate further development of NOD2 targeting agents.

Discovery and characterization of a high-affinity and high-specificity peptide ligand LXY30 for in vivo targeting of α3 integrin-expressing human tumors.

BACKGROUND: α3ß1 integrin is overexpressed in several types of human cancer and is associated with poor prognosis, metastasis, and resistance to cancer treatment. We previously identified a cyclic peptide ligand LXY1 that specifically binds to the α3ß1 integrin on human glioblastoma U-87MG cells. Here, we optimized LXY1 through one-bead one-compound combinatorial library screening and site-specific modifications to improve its in vivo binding property. METHODS: Three bead libraries were synthesized and whole-cell binding assays were performed. The binding capacity of individual peptide ligands against different tumor cells was determined by flow cytometry and confirmed by optical imaging. A complex joining biotinylated ligand with streptavidin-Cy5.5 was used for in vivo target imaging in both subcutaneous and orthotopic U-87MG xenograft mouse models. RESULTS: LXY30, a cyclic peptide with the sequence cdG-Phe(3,5-diF)-G-Hyp-NcR, emerged as the most potent and selective ligand for the α3 subunit of α3ß1 integrin with improved in vitro and in vivo tumor-targeting effects compared to LXY1 in U-87MG cells. LXY30 is considerably stable in plasma as demonstrated in an in vitro stability study in 90 % human plasma. LXY30 also binds to several other known α3ß1 integrin-expressing glioblastoma, lung, and breast cancer cell lines with various affinities. CONCLUSIONS: Our data support further investigating the role of LXY30 as a human tumor-targeting peptide ligand for systemic and intracranial delivery of imaging agents and cancer therapeutics.