Biology subjects

Alamgir, A.

Publications and source records attributed to Alamgir, A..

3 recordsLinked to original sources

bioRxiv2024

Programmable protein degraders enable selective knockdown of pathogenic β-catenin subpopulations in vitro and in vivo

Aberrant activation of Wnt signaling results in unregulated accumulation of cytosolic {beta}-catenin, which subsequently enters the nucleus and promotes transcription of genes that contribute to cellular proliferation and malignancy. Here, we sought to eliminate pathogenic {beta}-catenin from the cytosol using designer ubiquibodies (uAbs), chimeric proteins composed of an E3 ubiquitin ligase and a target-binding domain that redirect intracellular proteins to the proteasome for degradation. To accelerate uAb development, we leveraged a protein language model (pLM)-driven algorithm called SaLT&PepPr to computationally design "guide" peptides with affinity for {beta}-catenin, which were subsequently fused to the catalytic domain of a human E3 called C-terminus of Hsp70-interacting protein (CHIP). Expression of the resulting peptide-guided uAbs in colorectal cancer cells led to the identification of several designs that significantly reduced the abnormally stable pool of free {beta}-catenin in the cytosol and nucleus while preserving the normal membrane-associated subpopulation. This selective knockdown of pathogenic {beta}-catenin suppressed Wnt/{beta}-catenin signaling and impaired tumor cell survival and proliferation. Furthermore, one of the best degraders selectively decreased cytosolic but not membrane-associated {beta}-catenin levels in livers of BALB/c mice following delivery as a lipid nanoparticle (LNP)-encapsulated mRNA. Collectively, these findings reveal the unique ability of uAbs to selectively eradicate abnormal proteins in vitro and in vivo and open the door to peptide-programmable biologic modulators of other disease-causing proteins.

bioengineering↗

bioRxiv2024

Nanoparticle-mediated delivery of peptide-based degraders enables targeted protein degradation

The development of small molecule-based degraders against intracellular protein targets is a rapidly growing field that is hindered by the limited availability of high-quality small molecule ligands that bind to the target of interest. Despite the feasibility of designing peptide ligands against any protein target, peptide-based degraders still face significant obstacles such as, limited serum stability and poor cellular internalization. To overcome these obstacles, we repurposed lipid nanoparticle (LNP) formulations to facilitate the delivery of Peptide-based proteolysis TArgeting Chimeras (PepTACs). Our investigations reveal robust intracellular transport of PepTAC-LNPs across various clinically relevant human cell lines. Our studies also underscore the critical nature of the linker and hydrophobic E3 binding ligand for efficient LNP packaging and transport. We demonstrate the clinical utility of this strategy by engineering PepTACs targeting two critical transcription factors, {beta}-catenin and CREPT (cell-cycle-related and expression-elevated protein in tumor), involved in the Wnt-signalling pathway. The PepTACs induced target-specific protein degradation and led to a significant reduction in Wnt-driven gene expression and cancer cell proliferation. Mouse biodistribution studies revealed robust accumulation of PepTAC-LNPs in the spleen and liver, among other organs, and PepTACs designed against {beta}-catenin and formulated in LNPs showed a reduction in {beta}-catenin levels in the liver. Our findings demonstrate that LNPs can be formulated to encapsulate PepTACs, thus enabling robust delivery and potent intracellular protein degradation.

bioengineering↗

bioRxiv2024

Bioreversible anionic cloaking enables intracellular protein delivery with ionizable lipid nanoparticles

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic {beta}-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point towards a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

bioengineering↗