SNACIP: small molecule-nanobody conjugate induced proximity controls intracellular processes and modulates endogenous unligandable targets

Induced proximity can be used to control diverse cellular processes. We develop nanobody-based proximity inducers called SNACIPs, which can be used to regulate either tagged or endogenous proteins and demonstrate their use in blocking microtubule nucleation for tumor growth inhibition in vivo.
Published in Chemistry
SNACIP: small molecule-nanobody conjugate induced proximity controls intracellular processes and modulates endogenous unligandable targets

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Proximity-inducing mechanism orchestrates the proceeding of diverse cellular processes1. Chemically induced proximity or dimerization (CIP or CID) that uses a cell-permeable bivalent small molecule to bring two proteins in close proximity is a valuable strategy for regulating diverse biological processes, including signaling cascades, selective autophagy, axonal transport, cell therapeutic applications, and many others. However, CIP and CID technologies require genetic modification of cells, they are usually difficult to directly modulate unligandable and endogenous targets and the potential in therapeutic translation is practically restricted.

Microtubule nucleation is a biochemical even that initiates de novo formation of microtubules, a critical cellular process identified around 30 years ago2. Microtubule nucleation in spindle assembly is essential for maintaining life and the dysregulation of which is associated with many diseases, such as cancer3. It has been proposed that drugs that block microtubule nucleation would be better candidates than traditional microtubule targeting agents (MTAs). Traditional MTAs may have various sides effects, for example, interfering with neuronal functions that often leads to a condition known as chemotherapy-induced peripheral neuropathy (CIPN)4. Mechanistically, microtubule nucleation involves the concerted actions of large protein complexes and intrinsically disordered protein factors, all being rather challenging targets for small molecule drugs. On the other hand, these intracellular targets are also inaccessible by large molecular weight antibodies which usually cannot cross plasma membrane. 

In order to circumvent the limitations of traditional CIP systems, and to provide new biomedical insights in microtubule nucleation, we introduced small molecule-nanobody conjugate induced proximity (SNACIP) by integrating CIP, chemical nanobody engineering, and cyclic cell-penetrating peptide mediated non-endocytic delivery systems5. Nanobody is a camelid derived single variable domain of heavy chain of heavy-chain only antibody (VHH). Nanobodies are characterized by compact size, well solubility and superior stability, and compared to small molecules, nanobodies can be routinely generated with very high specificities and affinities toward their targets. Therefore, the possibility to use nanobody as one binding module of CIP can significantly expand the application potentials. Hence, we utilized a nanobody as one warhead to target a specific POI and a small molecule binding motif to induce proximity (Figure 1a). As small molecule-nanobody conjugates typically cannot easily cross the plasma membrane, we decided to modify the conjugate with a cyclic cell-penetrating peptide (cyclic CPP). Cyclic cell-penetrating peptide is a recently identified superior intracellular delivery cargo, which can efficiently mediate non-endocytic delivery of small-to-medium sized proteins. Therefore, we designed a cyclic decaarginine CPP, named Cys-cR10*, and found it to be an ideal cyclic delivery module6. Cys-cR10* is attached to the nanobody conjugate via a reductively cleavable disulfide bridge, and hence cR10* can be readily cleaved after entering the reducing intracellular environment, leaving behind the small molecule-nanobody conjugate for inducing proximity (Figure 1b).

Figure 1: Structural elements of SNACIP inducers and the working mechanism for inducing proximity inside live cells. a, General structural elements of SNACIP inducers in which the small molecule binding motif can be introduced either synthetically or posttranslationally. b, Schematic view of the structure and working mechanism of a representative SNACIP inducer, cR10*-SS-GBP-TMP (cRGT).

It is critical that SNACIP efficiently enters live cells in a non-endocytic fashion. Therefore, we studied the cellular entry mechanism using a fluorescein labeled SNACIP inducer. The SNACIP inducer starts to enter live Hela cells in a non-endocytic fashion within a few minutes and then smoothly distributed throughout the cell (Figure 2). We also found that the SNACIP inducer inside live cells shows no colocalization with endosomal puncta, validating the non-endocytic entry. Further, we found that the presence of an endocytic inhibitor either chlorpromazine or dansylcadaverine does not prevent the cellular entry of SNACIP.

Figure 2: Visualization of non-endocytic entry of a fluorescein labeled SNACIP dimerizer. a, Structural elements of Cys-cR10*. b, Visualization of the non-endocytic entry. c, Quantification of intracellular fluorescence enhancement along time (left) and line profile analysis (right) of the microscopic images shown in b.

We first designed a general purpose SNACIP inducer, cR10*-SS-GBP-TMP (cRGT), which features a trimethoprim (TMP) ligand for binding with Escherichia coli. dihydrofolate reductase (eDHFR), a green fluorescent protein (GFP) binding protein (GBP) nanobody, and a disulfide bridged cR10* cyclic CPP module. After cRGT enters a live cell, cR10* is released to liberate GBP-TMP to induce proximity for regulation of cellular processes. We first characterized cRGT in controlling of protein positioning. Hela cells co-expressing EGFP-mito (mito: mitochondria targeting sequence) and mCherry-eDHFR (cytosolic) were incubated with cRGT. We were happy to find that mCherry-eDHFR was recruited to mitochondria showing a high colocalization degree (Figure 3a). In view of these results, we then compared cRGT with several state-of-the-art CIPs including rapamycin (Rap), (+)-abscisic acid (ABA), and GA3-AM using the same translocation assay. According to the dose-dependent translocation curve, cRGT recruits cytosolic mCherry-eDHFR to mitochondria in a dose-dependent manner. Rap shows a “hook effect” which means that the optimal drug concentration is hard to control. cRGT achieves a larger possible translocation dynamic range than ABA. Compared to GA3AM, cRGT-induced dimerization can be readily reversed using TMP (Figure 3b). These results show the advantages of cRGT over other CIP systems.

Figure 3: The SNACIP inducer enables minute-scale, no-wash, reversible and dose-dependent control of protein positioning inside living cells. a, Translocation of mCherry-eDHFR from cytosol to mitochondria using cRGT. b, Comparison of cRGT with other state-of-the-art dimerizers using the translocation assay.

Endogenous intrinsically disordered proteins (IDPs) are challenging targets for traditional CIP and CID methods. Human TPX2 protein (hTPX2), a key microtubule nucleation factor, belongs to IDPs; and so far, no small molecule ligands that bind with hTPX2 have been reported, rendering TPX2 an valuable but unligandable target. Hence, we screened a nanobody against hTPX2 via phage display, and then designed latent type hTPX2 SNACIP inducers. Latent type SNACIPs utilize post-translational modification machinery to equip a small molecule binding motif, so that SNACIP inducers can also be used to control endogenous targets and without the need of genetic modification. The latent hTPX2 SNACIPs can undergo C-terminal farnesylation and then recruit endogenous hTPX2 to the proximity of the non-functional plasma membrane location. In this way, hTPX2 SNACIP drugs are designed and prepared that can off-regulate TPX2 functions (Figure 4a). According to in vivo evaluation results using xenograft mice model, we found that SNACIP inducers effectively inhibited tumor growth in vivo (Figure 4b). Hence, these results highlight the value of SNACIP technology for designing drugs that modulate endogenous intrinsically disordered unligandable targets.

Figure 4: hTPX2 SNACIP inducers effectively suppress hepatocarcinoma cell tumor growth in vivo. a, Structural element of cRTC and its worming mechanism. b, hTPX2 SNACIP inducer effectively inhibited tumor growth in vivo.

In summary, we introduced small molecule-nanobody conjugate induced proximity (SNACIP) as a valuable addition to currently existing sets of CIP/CID systems6. SNACIP inducers are able to directly modulate intracellular unligandable targets and endogenous intrinsically disordered proteins. We developed SNACIP drugs that inhibit microtubule nucleation for suppression of tumor growth in vivo, a topic that were unaddressed for around 30 years since the identification of microtubule nucleation in the middle of 1990th.

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  1. Stanton, B.Z., Chory, E.J. & Crabtree, G.R. Chemically induced proximity in biology and medicine. Science 359, 1-9 (2018).
  2. Moritz, M., Braunfeld, M.B., Sedat, J.W., Alberts, B. & Agard, D.A. Microtubule Nucleation by Gamma-Tubulin-Containing Rings in the Centrosome. Nature 378, 638-640 (1995).
  3. Draber, P. & Draberova, E. Dysregulation of Microtubule Nucleating Proteins in Cancer Cells. Cancers 13, 5638 (2021).
  4. Fukuda, Y., Li, Y.H. & Segal, R.A. A Mechanistic Understanding of Axon Degeneration in Chemotherapy-Induced Peripheral Neuropathy. Front. Neurosci. 11, 481 (2017).
  5. Herce, H.D. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nat. Chem. 9, 762-771 (2017).
  6. Sun, X., Zhou, C., Xia, S. & Chen, X. Small molecule-nanobody conjugate induced proximity controls intracellular processes and modulates endogenous unligandable targets. Nat. Commun. 14, 1635 (2023).

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