# Identifying the determinants of cell-penetrant miniproteins

> **NIH NIH F31** · NORTHWESTERN UNIVERSITY · 2023 · $42,907

## Abstract

Project Summary
Up to 75% of human-disease causing targets are considered “undruggable” using the current drug classes
available: small molecules and biologics. Many of these “undruggable” targets are involved in intracellular
protein-protein interactions (PPIs), which are challenging to inhibit with small molecules and antibodies. Small
molecules require hydrophobic pockets for binding, which PPIs generally lack. Proteins are capable of disrupting
PPIs, but they generally cannot cross the cell's plasma membrane. Miniproteins are small proteins (<10 kDa)
with defined tertiary structure. They are a promising class of protein therapeutics that have shown great success
at inhibiting extracellular targets and several cell-penetrant miniproteins have been identified. However, we still
do not know the design rules needed to develop miniproteins with intracellular delivery. The main bottleneck to
developing cell-penetrating miniproteins is that measuring cytosolic delivery is slow and expensive because each
protein must be purified and tested individually. To overcome this experimental limitation, we will develop an
entirely new multiplexed approach to measure the cytosolic delivery of many candidate cell-penetrant
miniproteins at once. We hypothesize that a high-throughput approach will rapidly uncover new cell-penetrant
miniproteins to study the determinants of cytosolic delivery. In Aim 1, we will develop a cytosolic capture and
pull-down approach to screen cell-penetrant miniproteins. We will create an entirely new approach that quantifies
the cytosolic abundance of each miniprotein in a mass spectrometry-based approach. The goal of this aim is to
extensively validate this method and see enrichment of our known cell-penetrant protein from a mixture of
inactive proteins added to cells. In Aim 2, we will define the protein features that contribute to the cytosolic
delivery of miniproteins. We will perform a second round of protein design to validate the features found in our
initial screen. The goal of this aim is to apply a multiplexed approach for measuring cytosolic delivery and identify
the protein features of successful cell-penetrant miniproteins. The long-term goal of the proposed work is to
determine the protein features needed for cytosolic delivery to develop new cell-penetrant miniproteins capable
of inhibiting PPIs implicated in disease.

## Key facts

- **NIH application ID:** 10752455
- **Project number:** 1F31GM151811-01
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Claire M Phoumyvong
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $42,907
- **Award type:** 1
- **Project period:** 2023-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10752455

## Citation

> US National Institutes of Health, RePORTER application 10752455, Identifying the determinants of cell-penetrant miniproteins (1F31GM151811-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10752455. Licensed CC0.

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