REPORT ON LIGAND- TARGET INTERACTION.

1.0 Introduction.

A protein–ligand complex is a complex of a protein bound with a ligand that is formed following
molecular recognition between proteins that interact with each other or with various other molecules.
Formation of a protein-ligand complex is based on molecular recognition between biological
macromolecules and ligands, where ligand means any molecule that binds the protein with high affinity
and specificity(Wikipedia,2023).

Protein-ligand interaction can also be said to be a database which describes the interactions which a
tiny molecules (identified by a Het_Id code) undergo with their respective target proteins, as observed
in PDB complexes (Google,2023).

Human carbonic anhydrase II (HCA II) is a monomeric zinc-containing metalloenzyme which speed up
the rate of hydration of carbon dioxide to the formation of bicarbonate and a positive ion(proton),an
equilibrium crucial to physiological processes such as respiration, pH balance,ion exchange, and bone
readsorption(Esbaugh et al,2006). (Fig1)

Fig. 1. Carbonic anhydrase catalyzes step 1 in a reversible manner. Step 2, the dissociation of carbonic
acid into bicarbonate and a proton, is a spontaneous process.

Presently, there are 16 human carbonic anhydrase isoforms known that differ in their interaction,
expression level, tissue distribution, and subcellular localization( Lehenkari,1998). Imtaiyaz et al,2013
stated that due to the catalytic isoforms of human carbonic anhydrase the active site is found at the
base of an ∼15 Å deep conical cleft, which is divided into two distinct hydrophilic and hydrophobic
layers that facilitate the transport of the bicarbonate ion and neutral carbon dioxide. The catalytic
domain contains a Zn2+ ion coordinated to three histidine residues and a nucleophilic hydroxide in a
tetrahedral coordination geometry. CO2 binds to a closer hydrophobic terminal, and an adjacent
threonine residue (Thr199) received a hydrogen bond from the Zn2+-coordinated hydroxide to activate
it for the nucleophilic attack,giving to the formation of bicarbonate.Given their crucial role in important
physiological processes, human carbonic anhydrases have been explored as pharmaceutical targets and
biomarkers for a variety of diseases (Aggarwa et al,2013).Carbonic anhydrase (CA) II is found in renal
tubules, brain, and osteoclasts, and is critical in acid-base homeostasis and bone remodeling (McMahon
et al., 2001; Lehenkari et al., 1998).
Amongst the human carbonic anhydrase isoforms, human carbonic anhydrase II (hCAII) is notably
expressed and found to be highly distributed in human tissues. As such, it has been explored as a target
in clinical treatments for indications including altitude sickness, edema,epilepsy, and glaucoma( Mishra
et al,2020). Defects in this enzyme are associated with osteopetrosis and renal tubular
acidosis(Lindskog,2017). Despite widespread clinical application for the treatment of glaucoma, there is
no first or second generation FDA-approved human carbonic anhydrase inhibitors that is isoform
selective. A consolidated efforts have tried to identify hCAII selective inhibitors that minimize off-target,
systemic activities to reduce the side effects and improve efficacy (Quigley,2011). Additionally, recent
studies have elucidated the important non-catalytic proton shuttling function of hCAII, which supports
lactate transport in cancer cells via its interaction with and subsequent activation of monocarboxylate
transporter isozyme1(Noor et al,2018). Such activity is the consequence of residues on the surface of
the enzyme and is thereby independent of enzymatic activity or inhibition (Becker,2020).

1.1 Structure.

Protein–ligand interactions are necessary to the most of the biochemical processes taking place in living
systems. Ligand-mediated signal transmission through the molecular pathway is essential to all life
processes; these biochemical interactions made up the biological recognition at the molecular stage.
The evolvement of protein function depends partly on the development of a highly specific sites
designed to bind small-molecule ligands with affinities tuned to the needs of the cell. Cooperativity in
ligand binding is very crucial to the controlling of the competing biological activities. Regulation of
cellular processes through a cooperative protein–ligand interactions occurs through molecular
mechanisms involving protein conformational transitions among low- and high-affinity states.
Concurrently, ligand-binding interactions are used to switch proteins among states of different function,
examples ranging from dioxygen transport to gene expression are presented. The structures of protein–
ligand complexes at atomic resolution make possible the design of small-molecular drugs for the
treatment of disease.(fig 2).

Fig 2.( a)Image from PDB showing the Protein-ligand interaction showing the Active site,bindi binding
site and the predicted regulatory ADP binding site.
1.2 Ligand Interaction.

Carbonic Anhydrase II in complex with Ferulic Acid

Fig 3. PDP DOI:6MBY

Carbonic anhydrase II in complex with carboxylic acid-based inhibitors.

Carbonic anhydrases (CAs) are molecular targets in various diseases. While many sulfonamide-based
drugs are in clinical use, CA inhibitor design is moving towards the incorporation of alternative zinc-
binding groups, such as carboxylic acids, to promote CA isoform-specific inhibition. Here, X-ray crystal
structures of CA II in complex with nicotinic acid and ferulic acid determined to 1.70 and 1.50 Å
resolution, respectively, are reported. Moreover, the structures of these two compounds are
superimposed with previously determined structures to compare the mechanisms of inhibition and the
properties of carboxylic acid-based CA inhibitors. The important class of alternative was examined ,non-
sulfonamide-based CA inhibitors and provides insight to improve the structure-guided design of CA
isoform-specific inhibitors ( Lomelino et al,2019).

Binding Affinity Annotations

ID Source Binding Affinity

FER BindingDB: 6MBY Ki: min: 2400, max: 2.10e+5 (nM) from 2 assay(s)

IC50: 6440 (nM) from 1 assay(s).

Carboxylic Acids

Carboxylic acids are a class of compounds that inhibit metalloenzymes through various mechanisms of
action which includes coordinating to the metal ion in a mono- or bidentate way. This is also the case for
CAs, for which a number of inhibition mechanisms have been noticed (Davis et al,2014). Firstly,
carboxylate compounds can directly bind to the catalytic zinc and displace the bound water/hydroxide
ion, as seen in classical sulfonamide inhibition (Figure 4) below.

Figure 4. (A) CA II (gray) in complex with cholic acid (yellow) (PDB:4PXX); (B) Surface representation of
CA II in complex with cholic acid (yellow),

Again, some carboxylates attach to the zinc-bound water/hydroxide ion through a hydrogen bond,
relating to the binding mechanism seen in phenol-based compounds (Figure 6). However, these
inhibitors are predicted to bind primarily in their anionic state ( Karioti et al,2015).

Figure 5. (A) CA II (gray) in complex with 2,6-dihydroxybenzoic (green, PDB:4E3F) [40]; (B) CA II (gray) in
complex with ligands (E)-3-(4-methoxyphenyl)but-2-enoic acid (magenta) and (E)-3-(4-chlorophenyl)but-
2-enoic acid (yellow) (PDBs: 5FNM and 5FLS, respectively)(Langella et al,2016) .(C) Overlay of carboxylic
acid–based compounds in CA II surface.

Finally, 1 carboxylate substituent was found to bind CA outside the active site, in a pocket adjacent to
the entrance (Figure 6). This binding blocks His64, the proton shuttle residue, in its “out” conformation,
leading to the inhibition of catalytic activity( Woods et al,2016).

Figure 6. The 2-[(S)-benzylsulfinyl] benzoic acid (periwinkle) in complex with CA II (gray) (PDB:4QY3)
(Woods et al,2015).

The moeity of carboxylic acid–based inhibitors can differ in both size and chemical properties, allowing
interactions with either the hydrophobic or hydrophilic half of the active site, in addition to isoform-
unique residues of the selective pocket. Again, the orientation of functional groups in relation to the
carboxylic acid ZBG has been shown to be an important factor in binding due to the possibility of steric
hindrance. Enhancing selectivity based upon the size of amino acids lining the active site cavity [46].
Compounds that extend further from the active site have been shown to increase the binding affinity of
carboxylic acid–based inhibitors by more than 100 fold, highlighting the importance of interactions with
the selective pocket residues, For instance, structures of CA II in complex with butenoic acid inhibitors
(PDB:5FNM and 5FLS) exhibit Ki values between 700–900 µM, while a more compact benzoic acid
derivative (PDB:4E3F) only has a Ki of approximately 5 mM. As these three compounds all anchor
through the zinc-bound water/hydroxide ion and form hydrogen bonds with conserved residue Thr199,
the additional van der Waals interactions formed by the butenoic acid derivatives with side chains in the
hydrophobic half of the active site dictate this increase in specificity (Figure 6).
Several other structures of carboxylic acid compounds have been found that exhibit isoform specificity.

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