Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells.

A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.

Mimicking kidney flow shear efficiently induces aggregation of LECT2, a protein involved in renal amyloidosis.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.

Loss of bound zinc facilitates amyloid fibril formation of leukocyte-cell-derived chemotaxin 2 (LECT2).

Aggregation of the circulating protein leukocyte-cell-derived chemotaxin 2 (LECT2) causes amyloidosis of LECT2 (ALECT2), one of the most prevalent forms of systemic amyloidosis affecting the kidney and liver. The I40V mutation is thought to be necessary but not sufficient for ALECT2, with a second, as-yet undetermined condition being required for the disease. EM, X-ray diffraction, NMR, and fluorescence experiments demonstrate that LECT2 forms amyloid fibrils in vitro in the absence of other proteins. Removal of LECT2's single bound Zn2+ appears to be obligatory for fibril formation. Zinc-binding affinity is strongly dependent on pH: 9-13 % of LECT2 is calculated to exist in the zinc-free state over the normal pH range of blood, with this fraction rising to 80 % at pH 6.5. The I40V mutation does not alter zinc-binding affinity or kinetics but destabilizes the zinc-free conformation. These results suggest a mechanism in which loss of zinc together with the I40V mutation leads to ALECT2.

Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients.

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998-1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients-including Tsc2-thereby preventing their ubiquitination and proteasomal degradation.

A Single Protein Disruption Site Results in Efficient Reassembly by Multiple Engineering Methods.

Disrupting a protein's sequence by cleavage or insertion of a hinge domain forms the basis for protein engineering tools, including fragment complementation, circular permutation, and domain swapping. Despite the utility of these designs, their widespread implementation has been limited by the difficulty in choosing where to interrupt the protein sequence: the resulting fragments often aggregate or fail to reassemble. Here, we show that an optimal site exists within ribose binding protein (RBP) that, when disrupted, results in the most efficient formation of fragment-complemented and domain-swapped species. Cleaving RBP at this site also produces a highly stable, cooperatively folded circular permutant. This hot-spot site was identified by an experimental approach involving selection among competing folds. We find that efficiency in the case of RBP is determined by kinetic factors (survival of the first) rather than thermodynamics (survival of the fittest). Together with emerging computational tools, this limited data set defines a pathway for designing robust platforms for molecular switches and biosensors based on the aforementioned protein modifications.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.

p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.

Converting a binding protein into a biosensing conformational switch using protein fragment exchange.

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.

Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein.

Alternate frame folding (AFF) is a protein engineering methodology the purpose of which is to convert an ordinary binding protein into a molecular switch. The AFF modification entails duplicating an amino- or carboxy-terminal segment of the protein and appending it to the opposite end of the molecule. This duplication allows the protein to interconvert, in a ligand-dependent fashion, between two mutually exclusive native folds: the wild-type structure and a circularly permuted form. The fold shift can be detected by placement of extrinsic fluorophores at sites sensitive to the engineered conformational change. Here, we apply the AFF mechanism to create several ribose-sensing proteins derived from Thermoanaerobacter tengcongensis ribose binding protein. Our purpose is to systematically explore the parameters of the AFF design. These considerations include the site of circular permutation, the length and location of the duplicated segment, thermodynamic and kinetic optimization of the switching mechanism, and placement of extrinsic fluorophores. Three of the four AFF variants created here undergo the expected conformational shift and exhibit a ribose-dependent fluorescence change. The fourth construct fails to switch folds upon addition of ribose, likely because the circularly permuted form folds much more slowly than the nonpermuted form. This disparity apparently introduces a kinetic barrier that partitions the refolding molecules to the nonpermuted structure. The results of this study serve as a guideline for applying the AFF modification to other proteins of biomedical, diagnostic, and industrial interest.

Engineering an artificial zymogen by alternate frame protein folding.

Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N- and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k(cat)/K(M) by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k(cat)/K(M) by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.

Enhancing response of a protein conformational switch by using two disordered ligand binding domains.

Introduction: Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Methods: Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and in vitro with purified protein. Discussion: Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.

Mimicking Kidney Flow Shear Efficiently Induces Aggregation of LECT2, a Protein Involved in Renal Amyloidosis.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of artery and capillary-sized channels-drives LECT2 fibrillogenesis. To mimic blood flow through the human kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering the mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

A generalizable nanopore sensor for highly specific protein detection at single-molecule precision.

Protein detection has wide-ranging implications in molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific interfaces for detecting proteins without the steric hindrance of the pore interior. Here, we formulate a class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. The outcomes of this work could impact biomedical diagnostics by providing a fundamental basis for biomarker detection in biofluids.

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.

A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro . The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

Protein conformational switches: from nature to design.

Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.

Engineering protein activity into off-the-shelf DNA devices.

DNA-based devices are straightforward to design by virtue of their predictable folding, but they lack complex biological activity such as catalysis. Conversely, protein-based devices offer a myriad of functions but are much more difficult to design due to their complex folding. This study combines DNA and protein engineering to generate an enzyme that is activated by a DNA sequence of choice. A single protein switch, engineered from nanoluciferase using the alternate-frame-folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for specific nucleic acid sequences, sensors for serotonin and ATP, and a two-input logic gate. nLuc-AFF is a genetically encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by a phone camera. nLuc-AFF retains ratiometric readout in 100% serum, making it suitable for analyzing crude samples in low-resource settings. This approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.

Engineering protein and DNA tools for creating DNA-dependent protein switches.

Switchable proteins are capable of changing conformations from inactive (OFF) to active (ON) forms in response to inputs such as ligand binding, pH or temperature change, or light absorption. A particularly powerful class of protein switches, exemplified by the Cas nucleases of CRISPR systems, are activated by binding of specific DNA or RNA sequences. The mechanism by which oligonucleotide binding regulates biological activity is complex and highly specialized in the case of Cas enzymes, but recent advancements in protein and DNA engineering have made it possible to introduce this mode of control into other enzymes. This chapter highlights recent examples of protein switches that combine these two fields of engineering for the purpose of creating biosensors that detect pathogen and other genomic sequences. One protein engineering method-alternate frame folding-has the potential to convert many proteins into ligand-activated switches by inserting a binding protein (input domain) into an enzyme (output domain). The steps for doing so are illustrated using GCN4 as a DNA recognition domain and nanoluciferase as a luminescent reporter that changes color as a result of DNA binding. DNA engineering protocols are included for creating DNA tools (de novo designed hairpins and modified aptamers), that enable the biosensor to be activated by arbitrary DNA/RNA sequences and small molecules/proteins, respectively. These methodologies can be applied to other proteins to gain control of their functions by DNA binding.

p53 and Zinc: A Malleable Relationship.

A large percentage of transcription factors require zinc to bind DNA. In this review, we discuss what makes p53 unique among zinc-dependent transcription factors. The conformation of p53 is unusually malleable: p53 binds zinc extremely tightly when folded, but is intrinsically unstable in the absence of zinc at 37°C. Whether the wild-type protein folds in the cell is largely determined by the concentration of available zinc. Consequently, zinc dysregulation in the cell as well as a large percentage of tumorigenic p53 mutations can cause p53 to lose zinc, misfold, and forfeit its tumor suppressing activity. We highlight p53's noteworthy biophysical properties that give rise to its malleability and how proper zinc binding can be restored by synthetic metallochaperones to reactivate mutant p53. The activity and mechanism of metallochaperones are compared to those of other mutant p53-targeted drugs with an emphasis on those that have reached the clinical trial stage.