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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5435_Библиотеки_им_академика_М_И_Перельмана
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These scales and their associated difficulties are succinctly outlined in the provided
diagram, encompassing a spectrum from the systemic level down to the molecular
level [19]. Traits of peptides that were once considered liabilities are no longer consid-
ered as problematic. For instance, injection is now considered an acceptable route of
administration for some indications, in part because longer-acting peptides or depot
formulations that require less frequent injections have been developed.
Additionally, in addition to historically dominating extracellular hormone recep-
tors, a variety of molecular targets are being addressed by peptide therapeutic candi-
dates. It is now possible to buy peptides that interfere with PPIs, target receptor
tyrosine kinases, and inhibit intracellular targets. New peptides have been identified
through phage display as the starting point for discovery and medicinal chemistry ac-
tivities, and unique peptide scaffolds have led to the development of new families of
peptide leads. A diverse and robust development landscape for therapeutic peptides
has resulted as a result of this. Over 150 peptide medications are currently undergoing
clinical studies, 260 more have undergone human clinical trials, and over 60 have al-
ready received regulatory approval in the USA, EU, and Japan.
14.3 Targeting the undruggable area with
therapeutic peptidomimetics
Protein phosphatases balance protein kinases’ enzymatic activity, so these two superfa-
milies play a key role in determining the status of protein phosphorylation, which can
change protein stability, macromolecular interactions, enzyme activity, subcellular local-
ization, and ultimately protein function, which regulates both healthy homeostasis and
disease processes, such as cancer. The 100 phosphatases, which dephosphorylate Ser,
Figure 14.1: Multiscale pharmacokinetics challenges (reproduced with permission from [19]).
354 Kannan Sadasivam, Venkata Surya Kumar Choutipalli, and Lalitha Gummidi
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Thr, or Tyr on protein substrates, appear to regulate the rate and duration of a signal,
providing an orthogonal mode by which cellular processes can be controlled, according
to mathematical modeling of the 500 kinases encoded in the human genome [20]. No
two classes better exemplify the overabundance and underabundance of drugs than kin-
ases and phosphatases, which play a crucial role in determining cancer cell survival and
response to medication therapy according to genetic research [21]. The discovery and
FDA approval of more than 35 protein kinase inhibitors, from the BCR/ABL inhibitor im-
atinib for chronic myelogenous leukemia in 2001 to the 2015 approval of Lenvatinib, a
multikinase inhibitor for thyroid cancer, and Palbociclib, cyclin-dependent kinase 4 and
6 inhibitors for breast cancer, represents one of the most remarkable achievements in
drug discovery [21].
Human Tyr phosphatases have received interest as prospective targets for cancer
therapy because aberrant Tyr phosphorylation plays a crucia l role in the develop-
ment and spread of cancer. Twenty-five of the 100 Tyr phosphatases have been found
to have gene amplification or overexpression in human cancer, which supports a role
in the etiology of the illness [22]. With 14 of the 25 potentially oncogenic phosphatases
shown to be downregulated, genetically deleted, mutated, or aberrantly spliced in
some human tumors, some Tyr phosphatases, like Ser/Thr phosphatases, are impli-
cated as tumor suppressors [22]. Conceptually, of course, this complicates the use of
any treatments because there is at least the theoretical chance that an inhibitor of
one of these phosphatases could have on-target effects that would increase rather
than decrease cancer. However, many of the available cytotoxic chemotherapeutics
raise these kinds of issues. The idea that Tyr phosphatases are an untreatable protein
class has been strengthened by attempts to find drugs that specifically block onco-
genic Tyr phosphatases using small chemicals, silencing RNA, or even antibodies.
These efforts have mostly failed to create a promising preclinical candidate.
The convergence of intracellular signaling mechanisms that result in gene expres-
sion is mediated by transcription factors (TFs). TFs are either directly or indirectly
engaged in a wide range of transcriptional and gene expression abnormalities that
are connected to cancer. Some of the first oncogenes discovered were TFs (c-MYC or
STAT3) or signaling pathway regulators that led to proximally increased transcription.
Consequently, there has been a lot of interest in creating methods to change these
transcriptional changes. TFs assemble protein complexes that are targeted at particu-
lar DNA locations. It has proven to be incredibly challenging up until recently to pre-
vent functional interactions between the nuclear TF protein and DNA or coregulatory
proteins with tiny compounds. In contrast to the deep, druggable binding pockets
found on the majority of enzymes or receptors, the sites on TFs engaged in these in-
teractions are typically broad, flat surface areas. In contrast to disrupting TF-DNA
binding, the best method currently known for modifying carcinogenic transcriptional
events with small compounds avoids interactions between proteins.
14 Computer-aided design of peptidomimetic therapeutics 355
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14.4 Modifying peptides and peptidomimetics
to target protein–protein interactions
The biological processes of cell division, signal transduction, transcription, translation,
and programmed cell death all depend on PPIs. PPIs also have a significant impact on a
number of diseases and pathological conditions including cancer, cardiovascular dis-
ease, and neurodegeneration [2 3]. It has been estimated that there will be roughly
650,000 PPIs in the interactome, which is the comprehensive list of molecular interac-
tions in a cell. PPI-modifying medicines provide enormous therapeutic potential since
PPIs mediate important molecular connections. Small compounds ar e typically em-
ployed to modify a specific enzyme function; however, targeting PPIs with these mole-
cules is difficult for two main reasons. First and foremost, there are numerous polar
and hydrophobic interactions present on the vast (1,500–3,000) binding surfaces be-
tween proteins. Additionally, binding surfaces are often flat and lack a clearly defined
binding pocket where a small-molecule medication could engage. Several recent re-
views [23, 24] have described the thrilling success stories and disappointing difficulties
that have resulted from targeting PPIs with small drugs.
On the other hand, peptides and peptidomimetics (modified peptides) are ideal
candidates to target PPIs. It’s interesting to note that a short linear peptide is thought
to mediate 15–40% of all PPIs (Figure 14.2) [24]. In addition, peptidomimetics can be
chemically changed to stabilize the bioactive conformation matching the 3D protein
structure. Peptides can be rationall y produced based on the natural sequences that
mediate PPI in proteins and, as a result, can hide a significant portion of the binding
surface. Additionally, peptides and peptidomimetics have the ability to modify intra-
cellular targets either independently crossing the cell membrane (like cyclosporine)
or by conjugating to peptides that c an penetratecells.Wewillgothroughtypical
methods for creating peptides to study and control PPIs in this article, along with in-
stances of peptides’ medicinal use as PPI regulators.
HTS is frequently used to find peptides that regulate PPIs. It is a common practice
to screen natural product libraries for PPI inhibitors since many naturally occurring
small compounds have evolved to interact with proteins and vice versa. Lar ge and
adaptable enough to modify protein surfaces, natural macrocyclic peptides in particu-
lar also make great therapeutic candidates due to their cyclic structure’simproved
stiffness, resistance to proteolysis, and cell permeability. For instance, robotnikinin, a
macrocyclic peptide that interferes with the interaction between the Patched and
Sonic hedgehog proteins in the hedgehog signal transduction pathway, was discov-
ered through screening of a 2,070-compound macrocycle library [25].
A peptide imitating the area mediating the PPI is a great place to start when creat-
ing inhibitors or instruments to study the interaction once that region has been iden-
tified. Short peptides are frequently fluid and unstructured, therefore stabilizing or
inducing the d esired secondary structure can boost the inhibitor’sefficacy.Alpha-
356 Kannan Sadasivam, Venkata Surya Kumar Choutipalli, and Lalitha Gummidi
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helices, B-strands, and loop or turn regions are the three primary protein structural
motifs that can be found at protein–protein interfaces; peptides that replicate these
motifs can be stabilized in various ways. A single alphahelix with at least two “hot-
spot” residues frequently plays a key role in the interaction between two proteins
[26]. By changing the amino acid sequence to incorporate natural or synthetic resi-
dues with a high tendency for forming helices, such as alpha-aminoisobutyric acid,
short peptide analogues of alpha helices can be stabilized [26].
14.5 New developments in peptide synthesis
The initial step in the creation of a peptide drug is the identification of prospective
therapeutic peptides. This is followed by chemical or biological peptide synthesis and
sequence alteration to enhance pharmacological effects. Pept ide synthesis is an ex-
tremely well-developed chemical process, particularly Merrifield’s SPPS method from
1963 [27]. The methodology and synthetic materials of SPPS technology have already
undergone significant advancements, and it now plays a critical role in the manufac-
ture of contemporary peptides. By combining amino acid coupling and deprotection
in a single straightforward reactor, it makes peptide synthesis easier, which has fur-
Figure 14.2: Small molecule PPI (protein–protein interaction) inhibitors and their spatial overlap with
continuous dominant peptides at the interaction interface. (A) In the complex between Bcl-xL and a
peptide from Bak (1ysi), the crystal structure reveals the dominant peptide, which interestingly
corresponds to the inhibitor binding site. (B–F) These examples demonstrate nonobvious multiple epitope
interfaces, where the dominant peptide also overlaps with the small molecule inhibitor: (B) XIAP-Bir3
interaction with Casp9 (1tfq); (C) human papilloma virus (HPV) proteins E2–E1 interaction (1r6n);
(D) LEDGFIntegrase interaction (3lpt); (E) IL2/IL2Ra interaction (1m48); and (F) TNFa trimer disrupter (2az5)
(reproduced with permission from [24]).
14 Computer-aided design of peptidomimetic therapeutics 357
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ther led to the development of automatic peptide synthesizers. The crude peptides
produced by SPPS are more monotonous than those produced using recombinant
technology because they lack additional biological substances like enzymes, DNA and
RNA fragments, unrelated proteins, and peptide s. Additionally, since the contami-
nants in the finished SPPS product are primarily the result of insufficient or unin-
tended reactions during the synthesis phase, further purification is quite simple.
Genetic code expansion produces short proteins and peptides that have a short
half-life due to their poor pharmacokinetics, which include fast serum breakdown
and rapid elimination. One method for increasing the half-life of protein therapies is
by attaching a polymer. PEG is a nonbiodegradable, nontoxic, low-immunogenic poly-
mer that is created by repeatedly joining units of ethylene oxide. Proteins’ effective
molecular weight can be increased with PEGylation to decrease their renal clearance
by kidney filtration. Additionally, the PEG moiety can protect proteins from proteo-
lytic enzyme digestion by increasing steric hindrance, and it can aid in boosting ab-
sorption by making the target p rotein more water soluble. PEGylation is a com mon
method for changing therapeutic proteins because of these benefits, and it has been
successfully used since the 1970s to enhance protein therapies. There are additional
possible cand idates in clinical studies, and there are currently more than 10 PEGy-
lated protein therapeutics available on the market [28].
14.6 Backbone cyclic peptidomimetics’ situation
today and their use in drug development
The creation of PPI inhibitors has received a lot of attention lately. The interfaces of
PPIs are often dynamic and flat in contrast to the well-defined hydrophobic binding
pockets for small molecule medications, which has restricted the use of conventional
small molecule on PPI targets. However, while having a high binding affinity to PPIs,
macromolecules like monoclonal antibodies lack cell permeability. The ability of bio-
molecules to be developed into medications that target intracellular proteins has been
significantly slowed down by poor cellular permeability. The chemical space of pepti-
des, particularly cyclic peptides and peptidomimetics, has larger potentials and is de-
serving of research when compared to small-molecule medications and biomolecule
pharmaceuticals. Peptides typically have spherical or disk-shaped three-dimensional
structures, which can interact with greater protein surface surfaces. Peptides are pos-
siblePPImodulatorsbecausetheyhaveincreased targeted specificity and affinity.
Peptides, particularly cyclic peptides, are useful building blocks for PPI targeted medi-
cation development because they maintain many characteristics of small molecules
including as stability and a lower likelihood of an immune response. Currently, there
are more than 60 peptide medications available for the treatment of infectious and
inflammatory illnesses as well as malignancies (Figure 14.3) [29].
358 Kannan Sadasivam, Venkata Surya Kumar Choutipalli, and Lalitha Gummidi
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In general, macrocyclic skeletons are preferable to their linear counterparts and have
garnered a lot of interest from both academia and business. Many different structural
alterations have been created in the realm of cyclic peptide drug development to increase
bioavailability. In the realm of structural cyclic peptide modifications, N-methylation, iso-
steres of the amide bond, and -carbon alterations are regularly employed techniques. To
fine-tune the structural conformation of cyclic peptides, the backbone N-methylation is a
preferred technique among them [29]. Regiospecific N-methylation of the backbone
amide has three effects: (i) it adds more steric restrictions; (ii) it selectively breaks one
hydrogen bond with the original amide NH; and (iii) it reduces the energy required to
change the amide bond’s conformation from cis to trans or vice versa. Amidol molecules
can assume cis conformations far more easily when they are N-methylated than when
they are unmethylated normally. Peptide macrocycles can automatically adopt conforma-
tions in vitro or in vivo, thanks to selective backbone amide N-methylations, which are
crucial for preserving the target molecules and enhancing membrane permeability [30].
14.7 The pharmacodynamics and pharmacokinetics
of peptidomimetics
The best delivery method for treating eye diseases can be chosen based on the physi-
cochemical characteristics of drug molecules, proximity to the target tissue, ocular
pharmacokinetics, and patient convenience. We discuss numerous ocular therapeutic
delivery methods that target the retina in this area, along with its pharmacokinetic
obstacles.
Phase I, 50,
10%
Withdrawn
8, 2%
Approved,
60, 12%
Discontinued,
261, 54%
Active, 155,
32%
Phase II, 75,
16%
Phase III, 25,
5%
Preregistered,
5, 1%
Figure 14.3: Development status of therapeutic peptides can be categorized as follows, with the numbers
indicating the count and percentage of peptides in each category (reproduced with permission from [29]).
14 Computer-aided design of peptidomimetic therapeutics 359
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Oral administration in the form of a tablet or liquid consumable or parenteral
routes, such as intravenous, intramuscular, subcutaneous, and intradermal injection
in an injectable form, can both achieve systemic delivery. These approaches are un-
usual for drug delivery in ophthalmology due to low ocular bioavailability and sub-
stantial systemic exposure. Systemically in jected medications must pass through the
blood ocular barriers, which are made up of the blood aqueous barrier (BAB) and the
blood retinal barrier (BRB) in order to reach the retina. These defenses are crucial for
preserving ocular homeostasis and preventing the entry of infections and harmful
chemicals into the eye. The ciliary body’s nonpigmented epithelium, iris epithelium,
and iris endothelium form tight connections that make up the BAB. The aqueous hu-
mor’s chemical makeup is crucially maintained by these tight connections. Due to the
tight connec tions, the BRB, which is made up of the endotheliu m of the retinal
capillaries and RPE, severely restricts the distribution of medicines from the systemic
circulation to the retina. Drug molecules depend on passive diffusion to traverse
these membranes in the absence of active transport. It has been demonstrated that
BRB molecules larger than 2 nm are blocked by their tight connections [31]. Small mol-
ecule permeability is heavily influenced by a compound’s lipophilicity. Another obsta-
cle to systemic medication delivery is systemic metabolism [32]. The medicine must
undergo systemic metabolism and be exposed to hepatic enzymes in the liver before
it can reach the eye. The amount of medicati on that can enter the retina is signifi-
cantly decreased by this procedure.
Due to their target specificity, peptidomimetic medications have the potential to
treat a range of eye illnesses. Many misregulated proteases have been demonstrated to
contribute to a range of eye illnesses and are prospective targets for medication devel-
opment using proteomics and genomes investigations from patients. A relevant bio-
chemical assay is required to examine the impact of the medication on the protease’s
function following the identification and validation of a protein linked to a disease. For
proteases, this is easily accomplished by employing fluorescently labeled peptide sub-
strates. By screening candidate inhibitors using this assay, hits and lead-like compounds
can be found. Cellular and animal models can be created throughout this process for
more sophisticated testing. Pharmacokinetic information is acquired once a group of
lead-like compounds have been identified, and then these compounds can be evaluated
in cellular experiments. These substances are examined in animals following prelimi-
nary toxicity studies in order to gather in vivo pharmacokinetic data such as absorp-
tion, distribution, metabolism, and excretion (ADME). Only the top compound with the
necessary effectiveness and pharmacokinetic characteristics is advanced at this point
for testing in bigger animals. Due to their low structural complexity, small peptidomi-
metics can be advantageous in formulatio n, whereas large peptidomimetics, which
have more protein-like properties, must take activity loss from structural alterations
into account. Additional pharmacokinetic considerations such intracellular absorption,
endosome escape, and nucleus targeting must be taken into account for intracellular
retinal medicines that target cytosolic chemicals or genomes.
360 Kannan Sadasivam, Venkata Surya Kumar Choutipalli, and Lalitha Gummidi
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14.8 Peptides that penetrate cells
Polypeptides and oligonucleotides are typically regarded as having poor therapeutic
utility due to their low biomembrane permeability and their relatively quick break-
down. This is a barrier for both the pharmaceutical industry and biomedical research.
In fact, the ability to give large hydrophilic chemicals intracellularly would expand
the possibilities of altering biological targets. Prior to recently, it seemed impossible to
transport hydrophilic macromolecules into the cytoplasm and nuclear compartments
of living cells without rupturing the plasma membrane. Thus, the finding that a pep-
tide derived from the homeodomain of the TF encoded by the Antennapedia gene (cf.
below) translocates across the plasma membrane of live cells, probably without the
help of membrane proteins, and allows the intracellular transport of conjugated oligo-
peptides and oligonucleotides has opened up new avenues in biomedical research.
Delivering hydrophilic macromolecules across the blood-brain barrier is an even
more challenging but crucial endeavor. To get beyond this obstacle, several strategies
have been considered. However, they are all constrained by issues like low yield in
brain administration or effectiveness limited to a small sample of compounds. Recent
research, however, raises the possibility that blood-brain barrier-crossing macromole-
cules could be transported via cell-penetrating peptides (CPPs) [33]. Depending on the
viral strain, Tat is a transcription-activating factor that ranges in length from 86 to 102
amino acids and is essential for HIV replication. It is divided into three distinct func-
tional domains [34] including an acidic N-terminal portion crucial for transactivation,
a cysteine-rich DNA-binding region (22–37 amino acids) containing a zinc-finger motif,
and a basic region (49–58 amino acids), which is in charge of nuclear import. The pro-
tein’s Ca21-independent cell attachment may also be mediated by the later region.
14.9 Intracellular peptides as potential therapeutic
candidates
CPPs do not exhibit cell-type selectivity and can penetrate cells without the assistance
of a membrane receptor. They are short sequences of amino acids (10–30 residues),
frequently positively charged. As there appear to be a variety of different criteria that
can classify a peptide as “penetrating,” an agreement over what constitutes a CPP has
not yet been reached. When experiments on pAntp showed that the peptide could be
joined to a bioactive substance (creating a “conjugate”) and exploited to ensure its in-
tracellular distribution, the tremendous potential of CPPs was realized. It’s notewor-
thy that after internalization into a living neuron, the associated cargo (a protein
kinase C inhibitor) continued to operate. Preliminary in vivo tests employing Tat re-
vealed the potential of CPPs to function as vectors for therapeutically active macromo-
lecules. Tat was covalently bonded to beta-galactosidase, a macromolecule that was
14 Computer-aided design of peptidomimetic therapeutics 361
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previously thought to be impervious to cells. A cell-type independent method was
used to deliver beta-galactosidase to the cytoplasm of numerous tissues [35].
The cytoplasmic transport of macromolecules by peptides had not been accom-
plished until these experiments. Tat and pAntp have created the framework that
made it possible. The possibility that any molecule may be coupled to a CPP in order
to successfully deliver it intracellularly was an exciting idea. We still do not fully un-
derstand the process by which CPPs enter cells and move around inside them, though.
Their clinical use is now restricted by this.
14.10 Therapeutic applications of peptidomimetics
Pharmaceutical companies have historically shunned the idea of using peptides as ther-
apeutic agents due to a number of drawbacks, including size, which makes them highly
susceptible to peptidase degradation, the absence of efficient delivery methods, poor
transport properties through biologic membranes, low oral bioavailability, rapid excre-
tion, and poor target specificity due to the flexible nature of peptides [36]. However,
there has lately been a resurgence of interest in peptides and peptidomimetics as poten-
tial therapeutic agents due to advancements in processing technologies. This is mainly
because stability, transport, affinity profiles, and oral availability have all seen various
advances. The development of new adjuvant and carrier systems, the advent of differ-
ent delivery modalities, and the advancement of proteomics in uncovering many PPI
targets have all boosted interest in peptides and their mimics as possible therapeutic
medicines [37]. The majority of efforts to prevent cardiovascular illnesses focus on re-
ducing or eliminating the risk factors that cause them such as high blood pressure, obe-
sity, inflammation, hyperglycemia, and hypercholesterolemia. Wider research into new
therapy methods like peptides and their mimics has been motivated by limitations in
currently available device therapies and pharmacologic medications for CVD.
The delivery of peptide medications is one of the main issues with their utiliza-
tion. Injections continue to be the most popular method of administration. Oral deliv-
ery, however, would be preferred due to the high level of patient compliance, which
raises a drug’s therapeutic value. Therefore, increasing oral bioavailability from less
than 1% to at least 30–50% still presents a difficulty. Due to the encapsulation of pep-
tides in nanoparticles (such as liposomes, synthetic polymers, or fullerenes), which
protects the medicine from protease digestion until needed and hence increases sta-
bility, the development of orally and nasally active formulations has recen tly been
proposed (Figure 1 4.4). Utilizing protease inhibitors, absorption boosters, or conju-
gated molecules in combination with the peptide structure, such as antibodies to im-
prove targeting, carbohydrates to increase solubility, or lipids to enhance peptide
permeability, are other approaches being researched to overcome peptide barriers
[38, 39].
362 Kannan Sadasivam, Venkata Surya Kumar Choutipalli, and Lalitha Gummidi
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Figure 14.4: (A) Versatility of magnetic nanoparticles in biomedicine; (B) CNT functionalization for siRNA delivery; and (C) ambidextrous nature of QDs in
nanomedicine (reproduced with permission from [39]).
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