Introduction

Drug molecules bind to proteins in an aqueous environment. Before binding, polar groups on the drug surface, such as hydroxyl groups, carbonyl groups, ether oxygens, and heterocyclic nitrogens, interact with surrounding water molecules, while water molecules with different positions and stabilities may also be present within the protein binding site. When a drug enters the binding site, some water molecules are displaced back into the bulk solvent, some remain, and others stay between the drug and protein, forming hydrogen bonds with both.[1-3]

In the human ataxia telangiectasia and Rad3-related kinase (ATR)–Camonsertib (RP-3500) complex structure determined in 2025, researchers modeled two bound water molecules in the drug-binding region: one connected to the tertiary alcohol of Camonsertib and the other located between the oxabicyclic oxygen atom and Ser2305 of ATR.[1] This structure provides a concrete example of how a drug can “use water” to connect with a protein through specific functional groups at defined three-dimensional positions.

1. Water Molecules in Protein Binding Sites

1.1 A Drug Does Not Enter an Empty Binding Pocket

Proteins exist in aqueous solution, and their surfaces and some internal cavities are inherently in contact with water. Before a drug enters a binding site, polar groups of the protein may already form hydrogen bonds with water, while the polar groups of the drug itself are also hydrated.

Drug binding simultaneously alters three classes of interactions:

The pre-existing drug–water, protein–water, and water–water interactions are reorganized, while new direct drug–protein interactions or water-mediated interactions are established. [2-4]

1.2 Bound and Ordered Water Molecules Occupy Relatively Well-Defined Positions

In high-resolution protein–ligand structures, some water molecules can be modeled at specific positions. Such water molecules are commonly referred to as bound water or ordered water.

They are often located between polar protein groups, drug functional groups, or other water molecules and form relatively complete local hydrogen-bonding networks.

The principal information that structural data can provide is:

where a water molecule is located and which surrounding atoms exhibit geometries compatible with reasonable interactions with it.

A clearly resolved water molecule in a structure does not mean that retaining it will necessarily increase affinity; likewise, displacing a water molecule does not necessarily strengthen binding.[2-4]

2. Geometric Basis of Water-Mediated Hydrogen Bonds

2.1 Water Can Act as Both a Hydrogen-Bond Donor and Acceptor

The oxygen atom of a water molecule possesses lone pairs and can act as a hydrogen-bond acceptor in common protein–ligand systems, whereas the hydrogen atoms of its O–H bonds can act as hydrogen-bond donors. A single water molecule can therefore form hydrogen bonds with multiple surrounding polar groups and participate in a local hydrogen-bonding network.[6]

When a protein and a drug form a direct hydrogen bond, the interaction can be simplified as:

Protein–D–H ··· A–Drug

Here, D–H represents the hydrogen-bond donor and A represents the hydrogen-bond acceptor. In actual systems, the hydrogen-bond donor may instead be located on the drug side and the acceptor on the protein side.

When a water molecule lies between the protein and the drug, it can form hydrogen bonds with polar groups on both sides, thereby creating a water-mediated hydrogen bond. For example, one common donor–water–acceptor arrangement can be represented as:

Protein–D–H ··· O(H₂O)–H ··· A–Drug

In this arrangement, the water molecule acts as a hydrogen-bond acceptor on the protein-facing side and as a hydrogen-bond donor on the drug-facing side. Depending on the donor/acceptor properties of the protein and drug functional groups, water can also adopt other hydrogen-bonding arrangements.

Thus, in a direct hydrogen bond, polar functional groups on the protein and drug establish direct polar contact, whereas in a water-mediated hydrogen bond, at least one water molecule participates in the local hydrogen-bonding network between the protein and drug. Both modes of interaction can coexist within the same protein–drug complex.

2.2 Water Bridges Can Compensate for Differences in Distance and Directionality

Hydrogen bonds have defined distance and directional requirements. The presence of an oxygen, nitrogen, or hydroxyl group in a drug structure does not mean that the group will automatically form an effective hydrogen bond after entering a protein binding site.

For example, although a hydroxyl group in a protein and an ether oxygen in a drug both possess the capacity to participate in hydrogen bonding, direct interaction may be weak if the two groups are too far apart or are not appropriately oriented.

When a water molecule is located between them:

Protein–OH ··· H₂O ··· O–Drug

Because a water molecule is small and can adopt multiple hydrogen-bonding orientations, it can compensate for some differences in distance and directionality, enabling two polar regions to form a continuous interaction network.[2,3]

Water-mediated hydrogen bonds can therefore organize two polar regions that would otherwise be difficult to connect directly into a suitable three-dimensional hydrogen-bonding network.

3. Bound Water and Protein–Drug Binding Free Energy

3.1 Formation of New Hydrogen Bonds Requires Reorganization of Pre-Existing Solvation Interactions

A hydroxyl group in a free drug molecule may already form a hydrogen bond with water:

Drug–OH ··· H₂O

After entering a protein binding site, it may instead form:

Drug–OH ··· Protein

Alternatively, water may remain as an intermediate bridge:

Drug–OH ··· H₂O ··· Protein

When a polar group moves from an aqueous environment into a protein binding site, it loses some of its pre-existing hydration interactions. This process is known as desolvation.

If the newly formed protein–drug interaction or water-mediated interaction cannot adequately compensate for the desolvation penalty, adding a polar functional group will not necessarily favor binding.[2-4]

This is a common phenomenon in medicinal chemistry: adding only one oxygen or nitrogen atom to a two-dimensional chemical structure may increase activity, leave it largely unchanged, or markedly reduce it.

3.2 Enthalpy and Entropy Jointly Determine the Actual Contribution of Bound Water

Protein–ligand binding free energy is jointly determined by enthalpy and entropy and can be expressed as:

ΔG_bind = ΔH_bind − TΔS_bind

where ΔG_bind is the binding free energy, ΔH_bind is the binding enthalpy change, ΔS_bind is the binding entropy change, and T is the absolute temperature.

A bound water molecule that forms favorable hydrogen bonds with both the protein and drug can provide beneficial interactions. On the other hand, compared with bulk water, ordered water at a relatively fixed binding site generally has more restricted translational and rotational degrees of freedom.

Displacing a bound water molecule back into the bulk solvent likewise produces two types of changes: the water molecule gains greater freedom of motion, but pre-existing protein–water, drug–water, or water–water interactions may also be disrupted.[2-4]

The study by Matricon et al. further showed that changes in binding resulting from water displacement can have a pronounced enthalpy-driven component and that perturbation of one water site may also affect the surrounding hydration network.[2]

4. Two Bound Water Molecules in the Camonsertib–ATR Complex

Camonsertib (RP-3500) is an ATR inhibitor. In 2025, Wang et al. determined the cryo-electron microscopy structure of human ATR in complex with ATR-interacting protein (ATRIP) and RP-3500. The overall reconstruction of the RP-3500 complex had a resolution of 3.79 Å, while the local resolution of the catalytic core containing the active site approached 3 Å, providing a basis for structural analysis of RP-3500 binding and the local bound water molecules.[1]

RP-3500 occupies the ATP-binding site of ATR. The researchers modeled two bound water molecules near the drug, designated w1 and w2. These two water molecules connect different oxygen-containing functional groups of RP-3500 with local structural elements of ATR, forming two sets of water-mediated hydrogen bonds.[1]

Figure 1. Binding Mode of Camonsertib (RP-3500) in the ATR Active Site and the Two Bound Water Molecules

The green stick structure represents RP-3500, the dark gray structures represent neighboring ATR residues, and the cyan dashed lines indicate hydrogen bonds.

① a, b: Three-dimensional positions of RP-3500 in the ATR active site viewed from two directions;

② c: Two-dimensional representation of the major interactions between RP-3500 and surrounding residues;

③ d: Enlarged view of the regions containing the two bound water molecules w1 and w2. Red spheres represent bound water molecules; w1 is located near the tertiary alcohol of RP-3500, whereas w2 lies between the oxabicyclic oxygen and S2305.[1]

Image source: Wang G. et al. Science Bulletin, 2025, 70, 2137–2146, original article Fig. 4, page 2143. Licensed under CC BY 4.0.[1]

4.1 Tertiary Alcohol–w1–Catalytic Loop Water-Mediated Hydrogen Bond

Camonsertib contains a tertiary alcohol hydroxyl group. Bound water molecule w1 is positioned between this hydroxyl group and the ATR catalytic loop. The tertiary alcohol hydroxyl forms a hydrogen bond with w1, while w1 simultaneously forms a hydrogen bond with the catalytic-loop region containing L2482.[1]

The local connectivity can be represented as:

Camonsertib–OH ··· H₂O(w1) ··· ATR catalytic loop

L2482 is leucine, whose side chain is a hydrophobic alkyl group. The water-mediated hydrogen bond here involves the backbone region of the peptide chain containing L2482.[1] The original study also noted that the water molecule near the tertiary alcohol is likewise observed in the single-crystal X-ray structure of RP-3500 monohydrate.[1]

w1 therefore enables the tertiary alcohol of Camonsertib to establish a continuous polar connection with the ATR catalytic loop through one water molecule.

4.2 Oxabicyclic Oxygen–w2–S2305 Water-Mediated Hydrogen Bond

Camonsertib also contains an oxabicyclic structure, whose ether oxygen can act as a hydrogen-bond acceptor.

Bound water molecule w2 lies between this ether oxygen and ATR serine 2305 (Ser2305, S2305). The original study showed that w2 forms hydrogen bonds with both the S2305 hydroxyl group and the oxabicyclic oxygen.[1]

The local connectivity can be represented as:

Ser2305–OH ··· H₂O(w2) ··· O–Camonsertib

This arrangement enables the ether oxygen of the oxabicyclic group to establish a polar connection with S2305 through w2.

The two bound water molecules in Camonsertib therefore form two distinct water bridges:

Tertiary alcohol → w1 → ATR catalytic loop

Oxabicyclic oxygen → w2 → S2305

The two water molecules connect different drug functional groups with different protein structural elements and jointly participate in the local hydrogen-bonding network of RP-3500 within the ATR active site.[1]

5. Ligand-Based Modulation of an Ordered Water Site in the A₂A Adenosine Receptor

Using the complex structure of the A₂A adenosine receptor (A₂AAR) with adenosine as a basis, Matricon et al. selected an ordered water molecule within the binding pocket as a structural target for investigation. This water molecule occupies a small cavity and forms hydrogen bonds with adenine N3 and the ribose 2′-hydroxyl group of adenosine, as well as with another crystallographic water molecule.[2]

The researchers compared four ligand structures around the same water site: adenosine (1), which retains the complete water-mediated interaction; 3-deazaadenosine (2), in which N3 is replaced by carbon, removing one ligand–water hydrogen bond; compound 3, in which a methyl group is introduced at the corresponding position to investigate displacement of the water site; and compound 4, in which a hydroxymethyl group is introduced to displace the ordered water while allowing the hydroxyl group to assume part of the hydrogen-bonding role previously performed by the water molecule.[2]

Figure 2. Ordered Water in the A₂A Adenosine Receptor and Ligand Structural Modifications around the Water Site

Panel a shows the local structure of the A₂A adenosine receptor–adenosine complex. Adenosine is shown as orange sticks, with surrounding receptor residues including His264, Glu169, Asn253, Asn181, His250, Ser277, His278, and Tyr9 labeled. Black dashed lines indicate hydrogen bonds. Red spheres represent other crystallographic water molecules, while the cyan sphere represents the ordered water molecule investigated in this study.[2]

Panel b compares the four ligand structures. Compound 1 is adenosine; compound 2 is 3-deazaadenosine, in which the nitrogen at the N3 position is replaced by carbon, eliminating one hydrogen bond with the target ordered water; compound 3 contains a methyl group at this position and represents a water-site displacement design; compound 4 contains a hydroxymethyl group and represents a design in which the ordered water is displaced while the hydroxyl group supplements the local hydrogen-bonding interactions. The labels Interact, Displace, and Replace in the original figure correspond respectively to interaction with the ordered water, displacement of the water site, and replacement of the water site.[2]

Image source: Matricon P. et al. Chemical Science, 2021, 12, 960–968, original article Fig. 1, page 961. Licensed under CC BY 3.0.[2]

5.1 Retention of the Water Site and Reduced Affinity after N3 Deaza Substitution

The adenine N3 of adenosine forms a hydrogen bond with the target ordered water molecule. In 3-deazaadenosine (compound 2), this N3 atom is replaced by carbon, directly removing one ligand–water hydrogen bond.[2]

In previous experiments, the affinity of 3-deazaadenosine for the A₂A receptor was approximately 39 μM; for adenosine, an estimated Ki of approximately 45 nM was obtained under experimental conditions without adenosine deaminase. Because the two measurements were performed under different experimental conditions, the original study did not treat the numerical difference as a precise fold comparison of affinity.[2]

Molecular dynamics simulations showed that the ordered water occupied the corresponding water site in 100% of simulation snapshots for the adenosine complex. After N3 deaza substitution, the water molecule remained at the original site in approximately 78% of simulation snapshots for 3-deazaadenosine. Thus, removal of N3 reduced the ligand–water hydrogen bonding interaction but did not completely eliminate the water site.[2]

Subsequent energy analysis showed that the affinity difference between adenosine and 3-deazaadenosine involved multiple contributions, including receptor–ligand complementarity, ligand internal energy, and hydration of the free ligand. Changes in hydrogen bonding to the ordered water in the bound state were not the dominant energetic source of this affinity difference.[2]

Thus, the loss of one water-mediated hydrogen bond in a structure can coincide with a decrease in affinity, but the entire affinity change cannot be attributed to that single hydrogen bond.

5.2 3-Methyl Substitution as a Water-Site Displacement Design

Compound 3 was derived from 3-deazaadenosine by introducing a methyl group at the corresponding position. The methyl group was designed to extend into the space occupied by the target ordered water, thereby allowing the effect of water-site displacement on ligand binding to be examined.[2]

Experimentally, compound 3 did not show significant radioligand displacement at 10 μM and also produced a low level of activation in the A₂A receptor functional assay.[2]

The available data indicate that the 3-methyl water-site displacement design did not restore the high affinity of the 3-deazaadenosine scaffold.

5.3 Water Displacement and Water-Network Reorganization after 3-Hydroxymethyl Substitution

Compound 4 was likewise derived from the 3-deazaadenosine scaffold, with a hydroxymethyl group introduced at the corresponding position. This structure was designed to occupy the position of the target ordered water while using the hydroxyl group to assume part of the local hydrogen-bonding role previously performed by the water molecule.[2]

In molecular dynamics simulations, the target ordered water was completely displaced in the compound 4 complex, whereas the other major hydration sites retained distributions similar to those observed in the adenosine complex.[2]

Experimental results showed that compound 4 had an A₂A receptor binding Ki of 103 nM and behaved as a full agonist, with an EC₅₀ of 732 nM in the functional assay. Compared with 3-deazaadenosine and 3-methyl compound 3, the 3-hydroxymethyl substitution restored relatively high receptor affinity and functional activity.[2]

Further free-energy and hydration-network calculations showed that the affinity gain of compound 4 relative to 3-deazaadenosine was markedly enthalpy driven. Following displacement of the ordered water, surrounding water–water and protein–water interactions were reorganized, with water-network rearrangement making an important contribution to the overall enthalpy change.[2]

The four structures in the study therefore represent three different levels of water-site modification:

6. Significance of Bound Water for Molecular Design

Water molecules within protein binding sites occupy different local environments. Some participate in relatively stable hydrogen-bonding networks and can be retained as part of the local interaction network; other water sites can be occupied by drug substituents; still others can, after displacement by ligand functional groups, be converted into new sites of direct polar interaction.[2-5]

In specific molecular-design settings, the key question is not simply whether a water molecule “should be retained or expelled,” but rather to evaluate:

what new interaction network the drug–protein–water system will form after the water site is retained, perturbed, or displaced.

The Camonsertib–ATR complex provides an example of bound water participating in drug–protein recognition. The tertiary alcohol and oxabicyclic oxygen of Camonsertib participate in the local ATR hydrogen-bonding network through w1 and w2, respectively.[1]

The A₂A adenosine receptor study further shows that a water site can be actively modulated through drug structural modification: removal of one hydrogen bond to an ordered water can coincide with a marked reduction in affinity, although the decrease cannot be attributed entirely to that single hydrogen bond; simply displacing the water site with a methyl group did not restore relatively high affinity, whereas introducing a hydroxymethyl group that could supplement local polar interactions while displacing the water restored relatively high receptor affinity and functional activity.[2]

Based on the RP-3500 complex structure, Wang et al. further proposed that hydroxyl groups or other hydrogen-bond donor/acceptor groups could be introduced at positions corresponding to the two bound water molecules in an attempt to form new direct hydrogen bonds with nearby ATR residues.[1]

This proposal remains a subsequent molecular-design hypothesis derived from structural information. No experimental results have demonstrated that displacement of w1 or w2 will necessarily further improve the potency or selectivity of Camonsertib. New functional groups may also affect drug conformation, desolvation, steric complementarity, and the local hydration network and therefore still require validation through compound synthesis and experimental evaluation.[2-5]

At binding sites involving bound water, the roles of polar groups such as hydroxyl groups, ether oxygens, or heterocyclic nitrogens after entering a protein binding site should not be understood solely in terms of an isolated hydrogen bond. Instead, they should be analyzed within the three-dimensional recognition system formed jointly by the drug functional group, bound water, protein residues, and neighboring water network.

Accordingly, seemingly small chemical structural changes can sometimes produce marked differences in activity. Changing a single atom or functional group may simultaneously affect the hydration state of the drug, the positions of bound water molecules, the hydrogen-bonding network, local conformation, and contacts with the protein.[2-5]

7. Chemicals Related to the Study of Bound-Water-Mediated Hydrogen Bonds and Drug–Protein Recognition

Table 1. ATR Inhibitors and PIKK-Family Selectivity Control Compounds

Table 2. Morpholine, Pyrimidine, and Pyrazole Building Blocks for Hydrogen-Bonding Pharmacophores

Table 3. Oxetane and Oxabicyclic Three-Dimensional Oxygen-Containing Building Blocks

Note: The products listed above are representative Aladdin products relevant to scientific research. Specific applications should be determined according to the product specification, batch COA, and the intended reaction or evaluation system. Additional product specifications, grades, and COA information can be searched on the Aladdin website using the product name/CAS/catalog number.

References

[1] Wang G., Wang P., Zheng Z., et al. Molecular Architecture and Inhibition Mechanism of Human ATR-ATRIP. Science Bulletin, 2025, 70: 2137–2146. DOI: 10.1016/j.scib.2025.05.009.

[2] Matricon P., Suresh R. R., Gao Z.-G., Panel N., Jacobson K. A., Carlsson J. Ligand Design by Targeting a Binding Site Water. Chemical Science, 2021, 12: 960–968. DOI: 10.1039/D0SC04938G.

[3] Spyrakis F., Ahmed M. H., Bayden A. S., et al. The Roles of Water in the Protein Matrix: A Largely Untapped Resource for Drug Discovery. Journal of Medicinal Chemistry, 2017, 60(16): 6781–6827. DOI: 10.1021/acs.jmedchem.7b00057.

[4] Abel R., Young T., Farid R., Berne B. J., Friesner R. A. Role of the Active-Site Solvent in the Thermodynamics of Factor Xa Ligand Binding. Journal of the American Chemical Society, 2008, 130(9): 2817–2831. DOI: 10.1021/ja0771033.

[5] Darby J. F., Hopkins A. P., Shimizu S., et al. Water Networks Can Determine the Affinity of Ligand Binding to Proteins. Journal of the American Chemical Society, 2019, 141(40): 15818–15826. DOI: 10.1021/jacs.9b06275.

[6] Arunan E., Desiraju G. R., Klein R. A., et al. Definition of the Hydrogen Bond: IUPAC Recommendations 2011. Pure and Applied Chemistry, 2011, 83(8): 1637–1641. DOI: 10.1351/PAC-REC-10-01-02.

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