TECHNOLOGIES USEFUL FOR ASSESSING PERMEABILITY

BACKGROUND

The invention relates to the field of biology and chemistry, particularly cellular biology and biochemistry.

Over the past three decades, there has been increased understanding of intracellular mechanisms and how these mechanisms signal cells to act. For instance, intracellular signaling can rapidly transmit signals from the cell surface to the nucleus, and vice versa, resulting in cellular activities including cell proliferation, apoptosis, differentiation, and changes in gene expression. Because of this increased understanding, there has been an identification of intracellular molecules (e.g., proteins) that can be stimulated and/or inhibited to change the way the cell will act. Sometimes, the stimulation of an intracellular target can be the result of a mutation or other damage to that target. For example, mutations and overexpression of intracellular molecule β-catenin is associated with many cancers.

Although the importance of intracellular molecules in the signaling within a cell has been known from decades, it has been a challenge to identify agents that can specifically target a particular intracellular molecule to affect that molecule's behavior, and thus affect the cell's activity. One of the challenges to identifying such an agent has been that it is difficult to predict whether an agent that specifically binds to a particular target (e.g., the Ras protein) will be able to traverse the membrane of a cell in order to access the intracellular space where their target resides.

The cell membrane of a non-plant eukaryotic cell is sometimes called a plasma membrane. The cell membrane is a double layer of lipids that separates the interior of a cell from the extracellular environment (e.g., the interstitial space and the fluids that reside therein). The lipids in the cell membrane are typically phospholipids. A phospholipid has a hydrophilic (water-loving) phosphate head along with a hydrophobic (water-repelling) fatty acid tail. In the double layer, phospholipid molecules are arranged such that the hydrophobic tails point inward, and the hydrophilic heads face outward. This double layer of phospholipids is called a phospholipid bilayer (see FIG. 1). The cell membrane in FIG. 1 would encircle the cytoplasm of a cell on the intracellular side of the depicted membrane, with the extracellular side of the membrane labeled accordingly. A cell membrane is comprised of a phospholipid bilayer along with molecules, such as proteins, that pass through all or some of the double layer membrane. For example, some molecules, such as ion channels, span the entire membrane, which one part exposed to the interior of the cell and the other part exposed to the extracellular surface. Other molecules may be attached to and partially contained within the interior layer of the plasma membrane, but do not pass through the membrane and thus do not protrude into the extracellular space.

Given its complexity, it is difficult to know if a molecule will be able to traverse the cell membrane in order to get to an intracellular target that the molecule will specifically bind to.

Thus, there is a need to discover a method for identifying intracellular target-specific agents that are able to traverse a cell membrane to access the intracellular target.

SUMMARY

Among other things, the present disclosure provides technologies for assessing barrier-crossing (e.g., cell membrane crossing) by an agent. In some embodiments, the present disclosure provides technologies for assessing interactions of a first agent (e.g., a capture molecule as described herein) with a second agent (e.g., a candidate compound linked to a binding moiety as described herein). In some embodiments, a first agent, e.g. a capture molecule, is limited in a region (e.g., a first region as described herein) by a barrier (a membrane, a layer, etc. as described herein). In some embodiments, a first agent is within a cell. In some embodiments, a first agent is within a liposome. In various embodiments, a first agent does not cross a barrier, or at very low level, to come in contact with a first agent. In some embodiments, after crossing a barrier, an agent such as a second agent forms a complex with a first agent, in some embodiments, by covalent linking. Such complexes can be utilized, as in many traditional technologies, for assessing barrier-crossing of a second agent and/or interactions between a first and a second agents. In some embodiments, after crossing a barrier, an agent, e.g., a second agent, is converted into a corresponding product agent (e.g., a product agent of a second agent) which possesses different properties and/or structures that can be utilized for detection. Among other things, the present disclosure demonstrates that utilization of such product agents of second agents can greatly improved sensitivity, accuracy and/or efficiency of detection and/or assessment. In some embodiments, multiple agents (e.g., 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 150, 200 or more) can be assessed at the same time with high-throughput, in some embodiments administered at the same time optionally in a single composition, e.g., when combined with mass spectrometry described herein.

In some embodiments, a first agent is or comprises a capture molecule as described herein. In some embodiments, a first agent is a capture molecule as described herein. In some embodiments, a first agent, e.g., a capture molecule, is or comprises a macromolecule agent, e.g. a protein. In some embodiments, a first agent is or comprises a polypeptide moiety. In some embodiments, a polypeptide is an enzyme. In some embodiments, a polypeptide is a target of a binding moiety, e.g., a binding moiety of a second agent. In some embodiments, a first agent is or comprises a functional group optionally linked to the rest of the first agent through a linker. In some embodiments, a functional group is an amino acid residue, e.g., of a polypeptide (e.g., an acid amino residue such as Asp, Glu, etc.). In some embodiments, a first agent comprises a functional group and a linker. In some embodiments, a linker in a first agent is or comprises a chemically cleavable linker (CCL). Various first agents and compositions thereof are described herein. In some embodiments, a provided agent is a first agent.

In some embodiments, a second agent is or comprises a candidate compound as described herein. In some embodiments, a second agent is a candidate compound linked to a binding moiety as described herein. In some embodiments, a second agent comprises a binding moiety which can interact with a first agent. In some embodiments, a binding moiety binds to a first agent upon contact. In some embodiments, a binding moiety can form a covalent linkage with a first agent upon contact. In some embodiments, a binding moiety is or comprises a functional group and optionally a linker. In some embodiments, a binding moiety is or comprises a functional group and a linker. In some embodiments, a binding moiety is or comprises a functional group and optionally a linker. In some embodiments, a binding moiety is or comprises a functional group and a linker. In some embodiments, a linker, e.g., in a second agent, is or comprises a chemically cleavable linker (CCL). In some embodiments, a functional group in a second agent binds to a functional group in a first agent. In some embodiments, functional groups in a first and a second agents react with each other so that a first and a second agents are covalently linked. In some embodiments, a reaction between functional groups in a first and a second agent is a bioorthogonal reaction. Those skilled in the art reading the present disclosure will appreciate that various bioorthogonal reactions and/or functional groups can be utilized in accordance with the present disclosure. In some embodiments, a second agent consists of or comprises a scaffold agent moiety and a binding moiety. In some embodiments, a second agent consists of or comprises a scaffold agent moiety, a functional group and optionally a linker. In some embodiments, a second agent consists of or comprises a scaffold agent moiety, a second functional group and a linker. In some embodiments, a scaffold agent is or comprises a small molecule. In some embodiments, a scaffold agent is or comprises a polypeptide. In some embodiments, a scaffold agent is or comprises a stapled peptide. In some embodiments, a scaffold agent is or comprises a nucleic acid. In some embodiments, a scaffold agent is or comprises an oligonucleotide. In some embodiments, a second agent is prepared from a scaffold agent. In many embodiments, a second agent is not prepared from a scaffold agent. For example, in some embodiments, a scaffold agent is or comprises a peptide moiety, and its corresponding second agent is prepared by replacing at least one amino acid residues in a scaffold agent with an amino acid residue which comprises or can be connected to a functional group optionally through a linker during peptide synthesis. Various second agents and compositions thereof are described herein. In some embodiments, a provided agent is a second agent.

Linkers in various agents may independently be or comprise CCLs. In some embodiments, a CCL, e.g., which is or comprises —C(O)O—, is cleaved in a chemical condition, e.g., an alkaline condition. In some embodiments, cleavage includes catalytic cleavage (e.g., promoted by a metal catalyst), acidic cleavage (e.g., as shown in FIG. 8C by TFA or FIG. 8D by 10% formic acid), oxidation cleavage, reduction cleavage, light-promoted cleavage (e.g., cleavage provided by UV light), etc. In some embodiments, cleavage of a CCL in a first and/or a second agent is promoted by an enzyme. In some embodiments, a CCL is or comprises a moiety that is cleavable by an enzyme. In some embodiments, an enzyme is or comprise a protease. In some embodiments, an enzyme is or comprise a peptidase. In some embodiments, an enzyme is or comprise a hydrolase. In some embodiments, a CCL is or comprises an ester group that is cleavable by an esterase. In some embodiments, a CCL is or comprises a polypeptide moiety that is recognizable and cleavable by an enzyme. In some embodiments, a first agent comprises a CCL and a second agent does not. In some embodiments, a second agent comprises a CCL and a first agent does not. In some embodiments, both a first and a second agents independently comprise a CCL. In some embodiments, none of a first and a second agents comprises a CCL. In some embodiments, a CCL is or comprises —C(O)—O—. In some embodiments, a CCL is or comprises vicinal diols. In some embodiments, a CCL is or comprises —N═N—. In some embodiments, a CCL is or comprises —Ar—N═N—Ar—, wherein each Ar is independently an aromatic moiety. In some embodiments, a CCL is or comprises a CCL moiety as described in FIGS. 8A-8E. In some embodiments, a CCL is formed by a reaction of a first and second agent. For example, in some embodiments, a CCL is or comprises —C(O)—O— which is formed by a —COOH functional group (or a salt or activated form thereof) of a first agent with a functional group, e.g., a leaving group such as —Cl, of a second agent.

In some embodiments, a linker is a covalent bond.

In some embodiments, agents of the present disclosure form complexes when in contact. For example, in various embodiments, a first agent and a second agent can form a complex. In some embodiments, a first agent and a second agent are covalently linked. In some embodiments, a first agent and a second agent are not covalent linked but form a complex through non-covalent interaction. In some embodiments, a non-covalent interaction comprises an interaction of biotin with a biotin-binding entity, e.g., avidin, streptavidin, etc. In some embodiments, provided technologies comprises enrichment of such complexes. In some embodiments, enrichment of such complexes may improve detection of certain agents, e.g., agents comprising releasing moieties as described herein. In some embodiments, a complex comprises a CCL. In some embodiments, a complex comprises two CCLs. In some embodiments, a complex comprises a CCL in a first agent moiety. In some embodiments, a complex comprises a CCL in a second agent moiety. In some embodiments, a complex independently comprises a CCL in a first agent moiety and a second agent moiety.

As described herein, in various embodiments, an agent, e.g., a first agent, a second agent, etc., is transformed, e.g., after crossing a barrier (e.g., a layer such as cell membrane). In some embodiments, a first agent is transformed into a product agent. In some embodiments, a second agent is transformed into a product agent. In some embodiments, a product agent is a product agent of a first agent. In some embodiments, a product agent is a product agent of a second agent. In some embodiments, a product agent is formed by transforming one or more groups in a first and/or second agent.

In some embodiments, a product agent of a first agent is or comprises a moiety of a second moiety, e.g., comprising a reaction product moiety of functional groups of a first and a second moiety. In some embodiments, a product agent of a first agent has a different molecular mass from a first agent. In some embodiments, a product agent of a first agent has a different molecular mass from a first agent. In some embodiments, such difference is detected by mass spectrometry.

In some embodiments, a product agent of a second agent is or comprises a moiety of a first moiety, e.g., comprising a reaction product moiety of functional groups of a first and a second moiety. In some embodiments, a product agent of a second agent has a different molecular mass from a second agent. In some embodiments, a product agent of a second agent has a different molecular mass from a second agent. In some embodiments, such difference is detected by mass spectrometry. For example, in various embodiments, —Cl of a second agent is converted into —OH in a product agent.

In some embodiments, a product agent comprises a releasing moiety. In some embodiments, a product agent is formed by cleavage of a CCL in a complex as described herein. In some embodiments, a product agent is or comprises a first and/or second agent moiety after cleavage.

In some embodiments, a product agent, e.g., a product agent of a second agent, consists of or comprises a scaffold agent moiety as described herein, a releasing moiety, and optionally a linker. In some embodiments, a scaffold agent of a product agent shares a characteristic structural element (e.g., a scaffold, a peptide sequence, a staple, or one or more characteristic portions thereof, etc.) as that of a second agent. In some embodiments, a scaffold agent of a product agent shares a common scaffold as that of a second agent. In some embodiments, a scaffold agent of a product agent is the same as that of a second agent. In some embodiments, a releasing moiety differs from a function group. For example, in some embodiments, a releasing moiety of a product of second agent is different from a functional group of a second agent. In some embodiments, a releasing moiety is —OH. In some embodiments, a functional group of a second agent is —Cl. In some embodiments, a releasing moiety is or comprises a moiety of a first agent. In some embodiments, a releasing moiety is or comprises a moiety of a reaction product of a first and a second agents (e.g., a moiety of a product moiety of functional groups in a first and second moiety).

In some embodiments, a product agent of a second agent and a second agent shares the same scaffold agent moiety or a characteristic structural element, and optionally the same linker. In some embodiments, a scaffold agent is or comprises a peptide. In some embodiments, a scaffold agent is or comprises a stapled peptide.

Various staple peptides may be utilized in accordance with the present disclosure, e.g., stapled peptides described in US 8,957,026, WO 2005/044839, WO 2008/061192, WO 2008/095063, WO 2008/121767, WO 2010/011313, WO 2011/008260, WO 2012/174423, WO 2012/174409, WO 2014/197821, WO 2008/137633, WO 2009/042237, WO 2009/108261, WO 2010/042225, WO 2010/068684, WO 2010/148335, WO 2011/094708, WO 2012/006598, WO 2012/065181, WO 2012/142604, WO 2013/055949, WO 2013/102211, WO 2013/142281, WO 2014/144768, WO 2014/144148, WO 2014/151369, WO 2016/149613, WO 2017/004591, WO 2017/040323, WO 2017/040329, U.S. 8,592,377, 9,556,227, 10,301,351, 9,163,330, US 2018/010001, U.S. Pat. No. 9,617,309, US 2018/0009847, US 2015/0225471, U.S. 10,081,654, US 2019/0202862, WO 2014/201370, WO 2015/051030, US 10,533,039, US 2020/0247858, and WO 2020/041270, the stapled peptides and staples of each of which is independently incorporated herein by reference. In some embodiments, a stapled peptide comprises one and only one staples. In some embodiments, a stapled peptides comprises two or more staples. In some embodiments, a staple is a hydrocarbon staple. In some embodiments, a staple comprises a carbamate group. In some embodiments, a staple comprises an amine group. In some embodiments, a staple is bonded to a residue whose backbone comprises a cyclic structure, e.g., a proline residue. In some embodiments, a staple is bonded to an alpha-carbon of an amino acid residue. In some embodiments, a stapled peptide is a stitched peptide.

In some embodiments, a product agent of a second agent and a second agent comprises the same scaffold agent moiety, wherein the scaffold agent is a stapled peptide.

In some embodiments, a provided agent is or comprises a first agent. In some embodiments, a provided agent is or comprises a second agent. In some embodiments, a provided agent is or comprises a complex agent, e.g., of a first and a second agent. In some embodiments, provided agent is or comprises a product agent. In some embodiments, provided agent is or comprises a product agent of a first agent. In some embodiments, provided agent is or comprises a product agent of a second agent.

In some embodiments, linkers, functional groups, releasing moieties, etc., may be attached to scaffold agent moieties at various locations. For example, in some embodiments, a scaffold agent is or comprises a peptide, and they may be connected at N-terminus, C-terminus, side chain(s), and/or staple(s) (if stapled peptide).

As appreciated by those skilled in the art, provided technologies, e.g., agents, compounds, etc., may be provided in various forms including various salt forms, such as various pharmaceutically acceptable salt forms. In some embodiments, agents or compounds are provided as pharmaceutically acceptable salts.

In some embodiments, the present disclosure provides a method comprising detecting a product agent of an agent. In some embodiments, formation of a product agent comprises an agent crossing a barrier, e.g., crossing a barrier into a region where a product agent or a precursor thereof is formed. In some embodiments, the present disclosure provides a method, comprising detecting a product agent of an agent, or a precursor thereof, in a region, when an agent enters a region by crossing a barrier and forms a product agent of an agent, or a precursor thereof, in a region. In some embodiments, an agent is a second agent as described herein. In some embodiments, a barrier is or comprises a layer. In some embodiments, a barrier is or comprises a lipid layer. In some embodiments, a barrier is or comprises a bilayer. In some embodiments, a barrier is or comprises a phospholipid bilayer. In some embodiments, a barrier is or comprise a membrane. In some embodiments, a barrier is or comprise a cell membrane. In some embodiments, a region is within a cell. In some embodiments, an agent is a second agent, and a product agent is a product agent of a second agent. In some embodiments, a product agent of a second agent or a precursor thereof is formed at a first region, wherein a first region and a second region are separated by a barrier. In some embodiments, a first region comprises a first agent, e.g., a capture molecule, as described herein. In some embodiments, a first agent is absent from a second region. In some embodiments, a first and a second agent bind to each other in a first region to form a complex. In some embodiments, a first and a second agent react with each other in a first region to form a complex. In some embodiments, a first and a second agent are covalently linked in a first region to form a complex. In some embodiments, a precursor of a product agent, e.g., of a product agent of a first and/or a second agent is a complex. In some embodiments, cleavage of a complex forms a product agent of a second agent. In some embodiments, cleavage of a CCL forms a product agent of a second agent.

In some embodiments, complexes are enriched, e.g., through affinity purification as described herein. In some embodiments, enrichment improves detection of a product agent of a second agent. In some embodiments, production of a product agent is after enrichment of a complex. In some embodiments, product of a product agent is through chemical and/or enzymatic digestion of a complex to yield a product agent of a first and/or a second agent. In some embodiments, product of a product agent is through chemical (e.g., basic conditions as described herein) and/or enzymatic cleavage of a CCL to yield a product agent of a first and/or a second agent. For example, in some embodiments, enzymes such as esterases are utilized to cleave —CO(O)—. In some embodiments, useful enzymes are or comprises proteases, peptidases and/or hydrolases. In various embodiments, conditions utilized are effective in cleaving CCL and are mild enough to not significantly damage one or more other moieties, e.g., scaffold agent moiety, candidate compound moiety, etc. or characteristic portions thereof. In some embodiments, conditions are mild enough not to significantly damage stapled peptide moieties or characteristic portions thereof.

In some embodiments, a complex is detected and its levels assessed. In some embodiments, product agents, e.g., of first agents and/or second agents, are detected and their levels assessed. Particularly, in many embodiments, product agents of send agents are detected and their levels assessed. In some embodiments, barrier-crossing of agents is assessed by presence and/or levels of product agents (e.g., compared to those of reference agents). In some embodiments, an agent is determined to cross a barrier by the presence and/or above a certain level of its product agent. In some embodiments, a reference agent does not cross a barrier or cross at very low levels (a negative reference agent). In some embodiments, a reference agent cross a barrier at a high level (a positive reference agent). In some embodiments, relative permeability factor of an agent, which is calculated as (level/amount of product agent of an agent-level/amount of product agent of a negative reference agent)/(level/amount of product agent of a positive reference agent-level/amount of product agent of a negative reference agent), is utilized to assess permeability across a barrier, e.g., a membrane such as a call membrane. In some embodiments, product agent ratio, which is calculated as (level/amount of product agent of an agent)/(level/amount of product agent of a positive reference agent) is utilized to assess permeability across a barrier, e.g., a membrane such as a call membrane. In some embodiments, reference agents and agents to be assessed are administered separately, e.g., as exemplified herein, in some embodiments, they were administered in separate wells wherein references agents were not administered in the same wells. In some embodiments, reference agents and agents to be assessed are administered simultaneously, in some embodiments, optionally in the same composition. In some embodiments, reference agents and agents to be assessed contact the same barrier. In some embodiments, reference agents and agents, as exemplified herein, are administered in the same wells. In some embodiments, a system comprises an agent and a reference agent (positive or negative), and a barrier (e.g., cell membrane of a cell). In some embodiments, a system comprises an agent, a positive reference agent and a negative reference agent, and a barrier. In some embodiments, a system comprises an agent and a reference agent (positive or negative) and a plurality of cells. In some embodiments, a system comprises an agent, a positive and a negative reference agent, and a plurality of cells. In some embodiments, a positive reference agent crosses membrane of a plurality of cells. In some embodiments, cells comprise first agents that agents to be tested, as second agents, can bind to or react with as described herein. For example, in some embodiments, cells comprise capture molecules as described herein. In some embodiments, a system is or comprises a composition comprising components therein in a well of a plate as described herein.

As described herein, one advantage of certain provided technologies is that multiplexing assessment can be performed. In some embodiments, a plurality of agents, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500 or more compounds are assessed simultaneously in a composition (in some embodiments, administered as a single composition; in some embodiments, added as one or more separate compositions). In some embodiments, a plurality of agents are or comprises members of a library. In some embodiments, a library of agents may be assessed at the same time; in some embodiments, directly from synthesis without purification. As exemplified herein, in some embodiments, a plurality of agents can be assessed together in a well. As described herein, typically a positive and/or a negative reference agent is also administered. In some embodiments, agents of a plurality share the same binding moieties; in some embodiments, binding moieties of one or more agents in the plurality are different from one or more other agents. In some embodiments, agents of a plurality share the same functional groups; in some embodiments, functional groups of one or more agents in the plurality are different from one or more other agents. In some embodiments, agents of a plurality share the same linker; in some embodiments, linkers of one or more agents in the plurality are different from one or more other agents. In some embodiments, agents of a plurality bind to or react with the same type of agents (e.g., capture molecules as described herein); in some embodiments, they bind to or react with different types of agents (e.g., capture molecules as described herein). In some embodiments, reactions are the same; in some embodiments, reactions are different, for agents of the plurality. In some embodiments, product agents of agents of a plurality (e.g., from digestion/cleavage of complexes formed by agents of a plurality with other agents, e.g., capture molecules, or otherwise converted from agents of a plurality) share the same releasing moieties; in some embodiments, releasing moieties of one or more product agents of agents in the plurality are different from one or more other agents. In some embodiments, product agents of agents of a plurality share the same linkers; in some embodiments, linkers of one or more product agents of agents in the plurality are different from one or more other agents. In some embodiments, for each agent in a plurality, its molecular mass is different from a product agent of the agent. In some embodiments, each agent independently has a different molecular mass. In some embodiments, a product agent of each agent of a plurality independently has a different molecular mass. In some embodiments, differences in molecular mass may be utilized for assessing barrier crossing, e.g., through assessment of levels of product agents utilizing mass spectrometry.

In some embodiments, an agent, e.g., a first agent such as a capture molecule as described herein, comprises a tag. In some embodiments, complexes formed by such agent with another agent, e.g., a second agent such as a candidate compound linked to a binding moiety as described herein, comprises a tag. In some embodiments, a tag is or comprises a His tag (e.g., a hexahistidine tag), a GST tag, a FLAG tag, etc. Among other things, tags may be utilized to enrich complexes and/or agents, which can be utilized for production of product agents. As appreciated by those skilled in the art, enrichment of agents and/or complexes comprising tags can be performed using various technologies as described herein. For example, in some embodiments, an agent, e.g., a capture molecule such as a capture protein with a bound agent (e.g., a complex formed), can be enriched/purified from treated cells by lysing the cells, and then isolating a capture molecule complex through the use of an affinity tag, such as such as a hexahistidine tag, or a GST tag, or FLAG tag, etc, that is fused to a capture protein. As described herein, such a step can allow separation of a capture molecule complex from other contents of a cell and any non-captured agent, and can among other things, improve signal of an assay and/or reduce false positives from uncaptured agents or other cellular contents. A product agent can then be released from a purified capture molecule complex using various methods as described herein.

In some embodiments, the present disclosure provides a method for identifying one or more candidate compounds that traverse an cell monolayer, the method comprising:

providing cell monolayer comprising a first side and a second side, the first side defining a first region, said first region comprising one or more capture molecules;

adding a plurality of distinct candidate compounds, each distinct candidate compound attached to a binding moiety, to a second region defined by the second side of the cell monolayer, under conditions whereby each distinct candidate compound of the plurality traversing the cell monolayer enters the first region and forms a complex with a capture molecule in the first region via a covalent bond between a portion of the binding moiety attached to the distinct candidate compound and the capture molecule, wherein one or more complexes are formed;

disrupting the one or more complexes to create one or more distinct candidate compounds each attached to a releasing moiety, said releasing moiety different from the binding moiety; and/or

identifying the one or more distinct candidate compounds attached to the releasing moiety as being one or more candidate compounds that traverses an animal cell monolayer.

In some embodiments, the present disclosure provides a method for determining if a candidate compound traverses an animal cell monolayer, the method comprising:

providing a cell monolayer comprising a first side and a second side, the first side defining a first region, said first region comprising one or more capture molecules;

adding the candidate compound attached to a binding moiety to a second region defined by the second side of the cell monolayer, under conditions whereby the candidate compound traversing the cell monolayer enters the first region and forms a complex with a capture molecule in the first region via a covalent bond between a portion of the binding moiety attached to the candidate compound and the capture molecule;

disrupting the complex to create the candidate compound attached to a releasing moiety, said releasing moiety different from the binding moiety; and/or

identifying the candidate compound attached to the releasing moiety as being a compound that traverses an animal cell monolayer.

In an aspect, the invention provides a method for identifying one or more candidate compounds that traverse an animal cell membrane, the method comprising providing phospholipid bilayer comprising a first side and a second side, the first side defining a first region, said first region comprising one or more capture molecules; adding a plurality of distinct candidate compounds, each distinct candidate compound attached to a binding moiety, to a second region defined by the second side of the phospholipid bilayer, under conditions whereby each distinct candidate compound attached to the binding moiety traversing the phospholipid bilayer enters the first region and forms a complex with a capture molecule in the first region via a covalent bond between a portion of the binding moiety attached to the distinct candidate compound and the capture molecule, wherein one or more complexes are formed; disrupting the one or more complexes to create one or more distinct candidate compounds each attached to a releasing moiety, said releasing moiety different from the binding moiety; and identifying the one or more distinct candidate compounds attached to the releasing moiety as being one or more candidate compounds that traverses an animal cell membrane. In some embodiments, the identifying step identifies the amino acid sequence of the candidate compound. In some embodiments, the identifying step identifies the structure of the candidate compound.

In another aspect, the invention provides a method for determining if a candidate compound traverses an animal cell membrane, the method comprising: providing phospholipid bilayer comprising a first side and a second side, the first side defining a first region, said first region comprising one or more capture molecules; adding the candidate compound attached to a binding moiety to a second region defined by the second side of the phospholipid bilayer, under conditions whereby the candidate compound attached to the binding moiety traversing the phospholipid bilayer enters the first region and forms a complex with a capture molecule in the first region via a covalent bond between a portion of the binding moiety attached to the candidate compound and the capture molecule; disrupting the complex to create the candidate compound attached to a releasing moiety, said releasing moiety different from the binding moiety; and identifying the candidate compound attached to the releasing moiety as being a compound that traverses an animal cell membrane. In some embodiments, the identifying step identifies the amino acid sequence of the candidate compound. In some embodiments, the identifying step identifies the structure of the candidate compound.

In some embodiments of the various aspects of the invention, the phospholipid bilayer is contiguous. In some embodiments of the various aspects of the invention, the phospholipid bilayer is a cell membrane of an animal cell. In some embodiments, the first region is an interior of a liposome. In some embodiments, first region is a cytosol of the animal cell. The animal cell may be the cell of a vertebrate animal. In some embodiments, the vertebrate animal is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a mouse, rat, hamster, monkey, rabbit, or dog. In some embodiments, animal cell is the cell of an insect. In some embodiments, animal cell is the cell of a bird. In some embodiments, animal cell is the cell of a fish. In some embodiments, the animal cell has a nucleus.

In some embodiments, the phospholipid bilayer is planar.

In some embodiments of the various aspects of the invention, the method further comprises the step of disrupting the phospholipid bilayer so that the first region and the second region are combined to create a mixed region after complex formation in the first region.

In some embodiments, the binding moiety is larger in mass than the releasing moiety. In some embodiments, the binding moiety is smaller in mass than the releasing moiety.

In some embodiments, the releasing moiety is created by replacing at least one atom of the binding moiety with at least one different atom.

In some embodiments, the complex is disrupted by exposing the complex to an environment with a pH of 11.0 or higher. For example, the pH may be 11.5 or higher, or the pH may be 12.0 or higher.

In some embodiments, disrupting the one or more complexes is or comprises breakup of an intermediate and/or release of a product by a capture molecule. In some embodiments, such a process is automatically performed by enzymes.

In some embodiments, the identification of the candidate compound attached to the releasing moiety is by mass spectrometry analysis.

In various embodiments, the candidate compound comprises a peptide. In some embodiments, the peptide comprises, consists essentially of, or consists of an alpha helical turn. In some embodiments, the peptide further comprises a small molecule scaffold stabilizing an alpha helical turn in the peptide. In some embodiment, the peptide is synthetic. In some embodiments, the capture molecule comprises a linker comprising a chemically cleavable linker and a functional group, said functional group able to covalently bind to at least a portion of a binding moiety attached to a candidate compound.

In some embodiments, capture molecule comprises, consists essentially of, or consists of a mutant form of a haloalkane dehalogenase, said mutant form lacking a hydrolase activity. In some embodiments, the capture molecule forms a covalent bond with a group selected from the group consisting of a benzylguanine derivative and a O₂-benzylcystosine derivative. In some embodiments, the capture molecule comprises, consists essentially of, or consists of a mutant form of an O⁶-alkylguanine-DNA alkyltransferase.

In some embodiments, the method includes providing phospholipid bilayer comprising a first side and a second side, the first side defining a first region, said first region comprising one or more capture molecules; adding a plurality of distinct candidate compounds, each distinct candidate compound attached to a binding moiety, to a second region defined by the second side of the phospholipid bilayer, under conditions whereby each distinct candidate compound of the plurality traversing the phospholipid bilayer enters the first region and forms a complex with a capture molecule in the first region via a covalent bond between a portion of the binding moiety attached to the distinct candidate compound and the capture molecule, wherein one or more complexes are formed; disrupting the one or more complexes to create one or more distinct candidate compounds each attached to a releasing moiety, said releasing moiety different from the binding moiety; and identifying the one or more distinct candidate compounds attached to the releasing moiety as being one or more candidate compounds that traverses an animal cell membrane.

In some embodiments, the invention provides methods and reagents for identifying target-specific agents that are able to traverse the cell membrane.

Additional aspects and embodiments of the invention are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a phospholipid bilayer, showing how individual phospholipid molecules orient themselves such that their hydrophilic tails face one another and their hydrophilic heads face away from each other.

FIG. 2 is a drawing of a schematic showing a non-limiting example of how a non-limiting candidate compound that is a peptide can be generated and attached to a binding moiety.

FIGS. 3A-3C are drawings showing a non-limiting embodiment of the invention. As shown in FIG. 3A, a phospholipid bilayer separates a first region from a second region, where the first region contains a capture molecule and the second region contains a candidate compound attached to a binding moiety. In FIG. 3B, if the candidate compound is able to traverse the phospholipid bilayer, the binding moiety will covalently bind to the capture molecule. In FIG. 3C, this complex of capture molecule: binding moiety: candidate compound is disrupted, creating a candidate compound attached to a releasing moiety.

FIGS. 4A and 4B are drawings showing a non-limiting embodiment of the invention, where a phospholipid bilayer separates a first region from a second region, where the first region contains multiple capture molecules and the second region contains a plurality of distinct candidate compounds, each attached to a binding moiety. As shown in FIG. 4A, Candidate Compound A does not traverse the phospholipid bilayer while Candidate Compound B and Candidate Compound C do traverse the phospholipid bilayer, and the binding moieties attached to Candidate Compound B and Candidate Compound C bind to capture molecules to form complexes in the first region. In FIG. 4B, disruption of the complexes results in Candidate Compound B attached to the releasing moiety and Candidate Compound C attached to the releasing moiety.

FIGS. 5A and 5B are drawings depicting a non-limiting type of planar phospholipid bilayer that can be used within various embodiments of the invention. FIG. 5B is a magnification of the indicated section in FIG. 5A showing the phospholipid bilayer.

FIG. 6 is a drawing showing a non-limiting embodiment of the invention in which the phospholipid bilayer is spherical and forms a liposome with an internal region (the first region) and an external region (the second region). As shown in FIG. 6, the capture molecule is within the first region and the candidate compound attached to a binding moiety is in the second region.

FIGS. 7A-7B are drawings showing a non-limiting embodiment of a binding moiety and capture molecule. In FIG. 7A, the candidate compound (R) attached to a binding moiety reacts with a capture molecule, where the capture molecule is a HaloTag protein, forming a complex comprising the HaloTag protein, a portion of the binding moiety, and the candidate compound. As shown by the circled atoms in FIGS. 7A-7B, the Cl atom in the binding moiety is replaced by an ester link in the complex, which is then replaced by a hydroxyl group (—OH) on the releasing moiety following disrupted by base hydrolysis.

FIG. 8A-8E are drawings of non-limiting examples of chemically cleavable linkers (CCLs) (in addition to or instead of the ester bond depicted in FIGS. 7A-7B), that can be used in the various embodiments of the present invention for disrupting complexes of capture molecules and candidate compounds.

FIGS. 9A-9C are drawings showing a non-limiting embodiment of the invention, where the capture molecule comprises multiple components. The capture molecule is constructed by the covalent attachment of the HaloTag protein in the first region with the small linker containing functional group FG1 in the first region to create a capture molecule (see FIG. 9A). A candidate compound attached to a binding moiety containing functional group FG2 (which is complementary to FG1) that traverses the phospholipid bilayer enters the first region, where the functional group FG2 reacts with the functional group FG1 of the capture molecule to form a complex comprising the capture molecule, the candidate compound, and a product comprising one or more covalent bonds from the reaction of FG1 and FG2 (FIG. 9B). The complex is disrupted by chemically cleaving the CCL to produce the candidate compound attached to a releasing moiety (FIG. 9C).

FIG. 10 is a drawing showing a non-limiting embodiment of the invention, where each of the capture molecule and the binding moiety attached to the candidate compound bears a functional group, where the functional groups are complementary to each other.

FIG. 11 is a drawing showing some non-limiting functional groups that are complementary to one another and thus can be used as FG1 (as a portion the capture molecule) and/or FG2 (as a portion of the binding moiety attached to the candidate compound) within various embodiments of the invention.

FIGS. 12A-12B are drawings showing a non-limiting embodiment of the invention where the capture molecule includes a modification of a SNAP-tag protein. As shown in FIG. 10A, the capture molecule is created in the first region by the covalent attachment of the SNAP-tag protein to the small linker that enters the first region by traversing the phospholipid bilayer. A candidate compound attached to a binding moiety (that includes a second functional group (FG2)) traversing the phospholipid bilayer will enter the first region and covalently bind to the capture molecule via the FG2 group bonding to the FG1 group to form a complex, which is then chemically cleaved to create a candidate compound attached to a releasing moiety (see bottom of FIG. 10B).

FIG. 13 is a drawing showing a non-limiting embodiment of the invention where the capture molecule includes a modification of a CLIP-tag protein. A capture molecule is created by covalent binding of a CLIP-tag protein to small linker (e.g., a small molecule linker) that includes a functional group (FG1), a chemically cleavable linker (CCL), and a benzylcytosine group (thereby releasing a cytosine molecule). A complex can then be created by covalent binding of the capture molecule to a candidate compound attached to a binding moiety that includes a second functional group (FG2) that is complementary to FG1. Disruption of the complex by cleaving the CCL creates a candidate compound attached to a releasing moiety.

FIGS. 14A and 14B are bar graphs showing side by side comparisons of a cell membrane traversing compound, Compound 1, and Compound 11, which does not traverse the cell membrane, in NanoBRET (FIG. 14B) and mass spectrometry permeation (14A).

FIGS. 15A and 15B are drawings showing a non-limiting candidate compound of the invention, specifically the stapled peptide Compound 1 attached to: (a) a non-limiting binding moiety (FIG. 15A) and (b) a non-limiting releasing moiety (FIG. 15B).

FIG. 16A and 16B are mass spectra of the candidate compound attached to a binding moiety of FIG. 15A and the candidate compound attached to a releasing moiety of FIG. 15B, respectively.

FIGS. 17A and 17B are bar graphs showing the cytosolic exposure (i.e., cytosolic presence) of the Candidate Compounds 2-10, all of which are stapled peptides, normalized to positive control stapled peptide Compound 1 as determined following testing of each compound individually (FIG. 17A) or together simultaneously (FIG. 17B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Among other things, the present disclosure provides various technologies, e.g., agents, methods, complexes, etc. as described herein. In some embodiments, provided technologies are useful for assessing barrier-crossing by an agent. In some embodiments, provided technologies are useful for assessing permeability of an agent across a barrier, e.g., a cell membrane. In some embodiments, provided technologies are useful for assessing membrane penetration of an agent. In some embodiments, the provided technologies are useful for identifying agents, e.g., candidate compounds, that can cross a barrier, e.g., a cell membrane.

In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents, and/or agents of certain properties (e.g., cell permeability) within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

As used herein, “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

The term “peptide” as used herein generally refers to a polypeptide that is typically relatively short, for example having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids. In some embodiments, a peptide, e.g., a stapled peptide, has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.

In some embodiments, a length of a peptide, a polypeptide, or a characteristic portion thereof, is 5 or more amino acids. In some embodiments, a length is 6 or more amino acids. In some embodiments, a length is 7 or more amino acids. In some embodiments, a length is 8 or more amino acids. In some embodiments, a length is 9 or more amino acids. In some embodiments, a length is 10 or more amino acids. In some embodiments, a length is 11 or more amino acids. In some embodiments, a length is 12 or more amino acids. In some embodiments, a length is 13 or more amino acids. In some embodiments, a length is 14 or more amino acids. In some embodiments, a length is 15 or more amino acids. In some embodiments, a length is 16 or more amino acids. In some embodiments, a length is 17 or more amino acids. In some embodiments, a length is 18 or more amino acids. In some embodiments, a length is 19 or more amino acids. In some embodiments, a length is 20 or more amino acids. In some embodiments, a peptide or a characteristic portion thereof is stapled.

As used herein, the term “characteristic portion”, in the broadest sense, refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a “characteristic portion” of a protein or polypeptide or peptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide or a peptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., of a protein, polypeptide, peptide etc.) is one that, in addition to the sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance.

In some embodiments, a characteristic portion may be biologically active. In some embodiments, a characteristic portion of a substance differentiates the substance from other substances.

Certain Agents Useful as First Agents/Capture Molecules

In some embodiments, the present disclosure provides an agent which comprises an agent which can bind to a binding moiety as described herein. In some embodiments, such an agent is a first agent as described herein. In some embodiments, such an agent is or comprises a capture molecule as described herein. In some embodiments, such an agent is or comprises a protein or a polypeptide. In some embodiments, a protein or a polypeptide comprises a functional group that can react with another functional group, e.g., of a second agent as described herein. In some embodiments, a functional group is or comprises —COOH, or a salt or activated form thereof. In some embodiments, such a functional group may react with another functional group, e.g., comprising a carbon to which a leaving group is attached. In some embodiments, in a reaction —C(O)—O— displaces a leaving group. In some embodiments, a leaving group is —Cl. In some embodiments, a protein or polypeptide is or comprises dehalogenase. In some embodiments, a protein or polypeptide is or comprises a haloalkane dehalogenase. In some embodiments, —COOH of an acid residue serves as a functional group for reactions with other functional groups, e.g., those of other agents. In some embodiments, a protein or polypeptide is expressed in a cell.

In some embodiments, an agent consists of or comprises a scaffold agent moiety which is linked to a functional group optionally through a linker. In some embodiments, a linker is or comprise a CCL. In some embodiments, such a functional group can react with a functional group of another agent, e.g., a second agent. In some embodiments, a scaffold agent is or comprises a protein or a polypeptide. In some embodiments, such an agent is constructed by modification of a protein or polypeptide, e.g., one expressed in a cell, by reacting a protein or polypeptide with an agent comprising a functional group. For example, in some embodiments, a protein or polypeptide is or comprises a SNAP tag protein or a characteristic portion thereof, and a suitable reagent for introducing a functional group to a SNAP tag protein or a characteristic portion thereof is an agent as described herein in FIG. 12A. In some embodiments, such an agent has the structure of:

R^F-L-FG1,

or a salt thereof, wherein FG1 is a functional group, L is a linker, R^Fis

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or Cl.

In some embodiments, R^Fis —Cl. In some embodiments, R^Fis

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In some embodiments, such an agent has the structure of Cl-L-FG1 or a salt thereof, wherein FG1 is a functional group. In some embodiments, a linker is or comprises a CCL.

In some embodiments, each linker, e.g., L, is independently a covalent bond, or a bivalent optionally substituted, linear or branched C_1-30group comprising one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1-10 heteroatoms, heteroaromatic moieties each independently having 1-10 heteroatoms, or a combination of one or more of such moieties, wherein one or more methylene units of the group are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, a bivalent C_1-6heteroaliphatic group having 1-5 heteroatoms, —C≡C—, —N═N—, -Cy-, —C(R′)₂—, —O—, —S—, —S—S—, —N(R′)—, —Si(R′)₂—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —C(O)C(R′)₂N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, an amino acid residue, or —[(—O—C(R′)₂—C(R′)₂—)_n]—, wherein n is 1-20;

each -Cy- is independently an optionally substituted bivalent monocyclic, bicyclic or polycyclic group wherein each monocyclic ring is independently selected from a C_3-20cycloaliphatic ring, a C_6-20aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R′ is independently —R, —OR, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selected from C_1-20aliphatic, C_1-20heteroaliphatic having 1-10 heteroatoms, C_6-20aryl, C_6-20arylaliphatic, C_6-20arylheteroaliphatic having 1-10 heteroatoms, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or:

two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-20 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms; or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

In some embodiments, a heteroatom is an atom that is not hydrogen or carbon. In some embodiments, each heteroatom is independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, including oxidized forms thereof.

In some embodiments, L comprises a CCL. In some embodiments, L is —L¹—CCL-L²—, wherein each of L¹, CCL, and L²is independently a covalent bond, or a bivalent optionally substituted, linear or branched C_1-20(e.g., C_1-5, C_1-10, C_1-15) group comprising one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1-10 heteroatoms, heteroaromatic moieties each independently having 1-10 heteroatoms, or a combination of one or more of such moieties, wherein one or more methylene units of the group are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, a bivalent C_1-6heteroaliphatic group having 1-5 heteroatoms, —C≡C—, —N═N—, -Cy-, —C(R′)₂—, —O—, —S—, —S—S—, —N(R′)—, —Si(R′)₂—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —C(O)C(R′)₂N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, an amino acid residue, or —[(—O—C(R′)₂—C(R′)₂-)_n]—, wherein n is 1-20.

In some embodiments, L¹is bonded to —R^F. In some embodiments, L¹is bonded to —Cl. In some embodiments, L¹is a linear C_1-10bivalent alkylene group, wherein one or more methylene units is optionally and independently replaced with —CH₂—O—CH₂— or —O—. In some embodiments, L¹is a linear C_1-10bivalent alkylene group. In some embodiments, L²is a linear C_1-10bivalent alkylene group, wherein one or more methylene units is optionally and independently replaced with —CH₂—O—CH₂— or —O—. In some embodiments, L²is a linear C_1-10bivalent alkylene group.

As those skilled in the art will appreciate, various CCLs may be utilized in accordance with the present disclosure in various agents of the present disclosure. In some embodiments, a CCL is or comprises —C(O)O—. In some embodiments, a CCL is or comprises

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