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•Peptides are a highly versatile class of promising drug candidates.•Peptides can be both passively and actively cell permeable.•It is now possible to develop cell permeable ...peptides.•Artificial amino acid incorporation contributes to more drug-like peptides.•Optimisation of sequences can enhance protease resistance and residence time.
The ready availability of potent peptide binders for any desired target highlights their potential impact as therapeutic agents. Despite their versatility, however, peptides tend to display unfavourable pharmacological properties, such as low bioavailability, high renal clearance and proteolytic degradation rates, and low cell permeability. Fortunately, an increasing number of promising strategies to produce novel peptides and furnish pre-existing scaffolds with more drug-like properties are now becoming available. These strategies include incorporation of non-proteinogenic amino acids, tag appendage to existing peptides and grafting onto scaffolds already possessing desirable pharmacokinetic properties. As a consequence, a variety of promising bioactive macrocyclic peptides have recently been discovered highlighting the promise of this class of molecules as future medicines.
•Peptides now represent an expanding class of novel drugs.•They are ideal candidates for pooled library screening approaches.•Technologies such as SICLOPPS, phage display and mRNA display give access ...to tight binding peptides.•Genetic code reprogramming & chemical modification provide more natural product-like libraries.•Non-standard motifs such as cyclisation and backbone methylations can now be introduced.
From their early roots in natural products, peptides now represent an expanding class of novel drugs. Their modular structures make them ideal candidates for pooled library screening approaches. Key technologies for library generation and screening, such as SICLOPPS, phage display and mRNA display, give unparalleled access to tight binding peptides. Through combination with genetic code reprogramming and chemical modifications, access to more natural product-like libraries, spanning non-canonical peptide space, is readily achievable. Recent advances in these fields enable introduction of diverse non-standard motifs, such as cyclisation and backbone methylations. Peptide discovery platforms now allow robust access to potent, highly functionalised peptides against virtually any protein of interest, with typical binding constants in the nanomolar range. Application of these optimised platforms in a drug discovery setting has the potential to significantly accelerate identification of new leads.
Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic ...screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >10
rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.
Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a ...histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical N
-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BH
1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BH
1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BH
1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.
ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable ...efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.
Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization ...of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.
Abstract
The universal genetic code, which specifies the 20 standard amino acids (AAs), forms the basis for all natural proteins. Researchers have developed efficient and robust
in vivo
and
in vitro
...strategies to overcome the constraints of the genetic code to expand the repertoire of AA building blocks that can be ribosomally incorporated into proteins. This review summarizes the development of these
in vivo
and
in vitro
systems and their subsequent use for engineering of peptides and proteins with new functions.
In vivo
genetic code expansion employing engineered othogonal tRNA/aaRS pairs has led to the development of proteins that selectively bind small molecules, cleave nucleic acids and catalyze non‐natural chemical transformations.
In vitro
genetic code reprogramming using Flexizymes coupled with mRNA display has resulted in potent macrocyclic peptides that selectively bind to therapeutically important proteins. Through these examples, we hope to illustrate how genetic code expansion and reprogramming, especially when coupled with directed evolution or
in vitro
selection techniques, have emerged as powerful tools for expanding the functional capabilities of peptides and proteins.
The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology ...holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains
. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present
. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref.
) converts a de novo Diels-Alderase into a photoenzyme for 2+2 cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.
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