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Understanding the Electron Transfer Pathway of Cytochrome P450s: A Structural Point of View

WANG Yue, 2 March 2023

Cytochrome P450s are a large and ancient enzyme superfamily found in all kingdoms of life. They have a unique ability to detoxify chemicals in humans, making them an important target in medicinal and pharmaceutical drug discovery. The term “P450” is derived from the spectrophotometric peak at the wavelength of the absorption maximum of the enzyme (450 nm) when it is in the reduced state and complexed with carbon monoxide. The official nomenclature of P450 is based on gene sequence similarity, which assigns a family number (e.g., CYP1, CYP2), followed by a subfamily letter (e.g., CYP1A, CYP2B) and then several individual proteins (e.g., CYP1A1, CYP2B6).

P450 systems mainly consist of two functional parts: the heme-containing P450 domain and redox partners (RPs). The P450 domain contributes to substrate binding and transformation, while the RPs contain redox centers that relay electron equivalents from electron donors (usually NADPH) to activate the dioxygen bound to the P450 domain. Together, these events form an electron transfer (ET) pathway of NADPH-RPs-heme-O2.

Coacervates are condensed liquid-like droplets formed by liquid–liquid phase separation of molecules through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides can form simple and complex coacervates. This emerging peptide-based coacervate holds promise as efficacious delivery vehicles for multi-purpose biomedical applications.

The core region of the heme domain of all known P450s folds into a triangular globular domain (Figure 1A). Membrane-bound P450s have an additional N-terminal transmembrane domain and a flexible linker that may influence the localization and orientation in the membrane. The catalytic center of P450 is a Fe ion that is ligated to a protoporphyrin XI macrocycle and two additional axial ligands[1]. The proximal one is a thiolate of the strictly conserved Cys from the enzyme, and the distal one is a ligand that varies during the catalytic cycle. The elongated helix I running through the distal side of the heme contains residues that interact with substrate and dioxygen (Figure 1A). Helix F and G, which lie above helix I, are highly flexible and control the opening of the substrate entry tunnel.[2] The space of the closed-form pocket varies substantially in different P450 domains, determining the enzyme-substrate specificity. The heme-coordinating Cys residue located on the C-terminus of the helix L is situated on the proximal side of the heme group (Figure 1B). The proximal side is considered a portal to accept electrons delivered from RPs, and thus there are fewer structural elements on this side. The Cys sulfur is hydrogen-bonded to the nearby peptide NH groups to render the heme iron a sufficient redox potential.[3-4]

Because P450 domains share a similar fold, using the diverse RP systems used by various P450s is also a common approach to categorize these enzymes. Class II P450s are the most common eukaryotic P450s and are generally found in the endoplasmic reticulum (ER). These P450s employ a two-component system that comprises a membranous P450 domain and NADPH-cytochrome P450 reductase (CPR; Figure 2, class II). In the human liver, class II CYPs and CPRs are anchored on ER membrane by a single TM domain with their catalytic domain facing the cytosol. CPR contains FAD-containing reductase and FMN-binding domains that are linked via a “hinge” region, which is presumed to confer conformational plasticity of two flavin-binding domains.

Peptide-based liquid droplets as emerging delivery vehicles

CHEN Hongfei, 2 Feb 2023

Coacervates are condensed liquid-like droplets formed by liquid–liquid phase separation of molecules through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides can form simple and complex coacervates. This emerging peptide-based coacervate holds promise as efficacious delivery vehicles for multi-purpose biomedical applications.

1. Peptides as building blocks for liquid microdroplets
There are mainly four kinds of peptide-based coacervate: simple coacervate formed by peptides and peptide derivatives, polymers formed by repeated peptide motifs, complex of oppositely charged peptides and polyelectrolytes, and polymer–peptide hybrids (Figure 1). [1-4] Simple coacervation of peptides is formed by interactions of a single peptide or peptide derivative in aqueous buffer which often triggered by a change in temperature, pH or salinity. The microdroplets contain most of the peptides originally in solution and a significant amount of hydration water. [1] The engineering peptide polymers containing tandem repeats of sequences can form coacervate more easily. Compared with short peptides, peptide polymers are more prone to phase separation because their mixing entropy per amino acid is smaller. [2] By combining two oppositely charged peptides complex coacervates can be formed. Oligopeptides as short as ten amino acids can form stable salt-responsive complex coacervate microdroplets whose critical salt concentrations can be tuned over two orders of magnitude by changing peptide length and composition. [3] Besides of this, short peptides can be grafted onto a hydrophilic polymer backbone to enhance their hydration and create coacervates that more closely mimic the physicochemical properties of membraneless organelles. The hybrids phase separates into liquid microdroplets resembling membraneless organelles in vitro and in living cells. [4]

2. Peptide-based coacervates as stimuli-responsive delivery vehicles
Peptide-based coacervate droplets, which range from nanometers to micrometers, are promising delivery vehicles. Within the microdroplets, molecules can be hold by weak non-covalent interactions, allowing the packaging of different types of therapeutics. Compared with solid particles that are more commonly used for delivery, such as polymer, inorganic or lipid nanoparticles, coacervates are viscoelastic objects that can quickly recruit cargoes under aqueous conditions, which is especially beneficial for preserving the bioactivity of biomacromolecular therapeutics (Figure 2).

Coacervate, developed from thermo-responsive ELPs fused to therapeutics, can greatly prolong retention times of the therapeutics when injected subcutaneously or directly at the tumor sites. Rational molecular design can tune the coacervation temperature which enables the peptides to be injected as a solution at room temperature and then form viscous depots at body temperature. Using this concept, an ELP fused to glucagonlike peptide 1 (a promising drug for treatment of type 2 diabetes) was designed so that it was soluble at ambient temperature for ease of injection, but quickly transitioned at body temperature into a droplet with slow-releasing characteristics. With this strategy, the blood sugar level could be controlled for up to 17 days in a diabetic primate animal model. [5] Peptide microdroplets can also be used to deliver therapeutics inside the cell.

Introduction to Antibody-Drug Conjugates and Recent Advances in Linker

LI Biquan 22 Dec 2022

Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. ADCs combine the targeting properties of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, designed to discriminate between healthy and diseased tissue[1]. Totally, ADCs include three parts: antibody, linker and drug, as shown in Figure 1. Firstly, we could divide the conjugations between antibody and linker to site-specific conjugations and non-specific conjugations. On the other hand, linkers comprise two key parts: the antibody- and the payload-attachment (Figure 1). The bonding between the linker and the mAb is critical since it defines the drug-to-antibody- ratio (DAR, in generally 3 to 4 and maximum would not exceed 8 to 9) and therefore the homogeneity and stability of the ADC. The bonding between the linker and the payload is equally important and determine the releasing mechanisms of the drugs[2-4].

Additionally, we have to make a clear mind at the mechanisms that how ADCs enter the target cells. As shown in Figure 2 (A), endocytosis leads ADCs to the lysosome, a unique compartment with a low pH and high concentration of hydrolytic enzymes. Alternatively, ADC linkers can also be labile to the extracellular tumor microenvironment, according to the non-internalizing mechanism of action previously discussed (Figure 2 (B))[2-3].

For ADCs to be selective and potent, the linker technology employed should strive towards three key properties: (1) High stability in circulation. (2) High water solubility to aid bioconjugation and avoid formation of inactive ADC aggregates. (3) Allow efficient release of a highly cytotoxic payload-linker metabolite[3]. ADC linkers can be classified as ‘cleavable’ or ‘non-cleavable’. For non-cleavable linkers, there is no inbuilt chemical trigger to cleave the linker. Conversely, cleavable linkers exploit specific conditions to release the drug at the target cell. More than 80% of the clinically approved ADCs employ cleavable linkers. In addition, cleavable linkers can be further subcategorized as chemically- cleavable or enzyme-cleavable[3].

Chemically cleavable linkers could be subcategorized as acid cleavable, reducible disulfides and those cleavable by exogenous stimuli, such as extra adding chemicals and photo triggers. On the other hand, enzyme cleavable linkers so far include cathepsins cleavable, glycosidase cleavable sulfatase cleavable and phosphatase cleavable linkers[3-4].

1 Chemically Cleavable Linkers

Acid cleavable linkers aim to exploit the acidity of the endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), whilst maintaining stability in circulation at pH 7.4. This strategy yielded the earliest clinical success in the field with Pfizer’s gemtuzumab ozogamicin (Mylotarg) ADC and was later employed in Besponsa[5-6] (Shown in Figure 3). In 2019, the group[7] developed a novel silyl ether-based acid-cleavable ADC carrying highly cytotoxic monomethyl auristatin E (Shown in Figure 3). Glutathione (GSH)-cleavable (reducible disulfides) linkers rely on the higher level of glutathione in the cytoplasm (1-10 mmol/L) compared to the blood plasma (5 mmol/L)[8]. Disulfides are the most prominent class of chemically cleavable motifs found in ADC linkers and, alongside hydrazones, they have found clinical success in Pfizer’s Mylotarg and Besponsa. Disulfides are stable at physiologic pH but are susceptible to nucleophilic attack from thiols (Figure 4) [3-4]. The examples are shown in Figure 4. Whilst the cleavage of ADC linkers with endogenous triggers is the simplest method for drug release, external small molecule triggers of extracellular drug release may offer the following advantages: (1) avoidance of any disparity in linker cleavage rates caused by variable biology across patients, and (2) the ADCs can be effective when extracellular concentrations of endogenous triggers are insufficient for efficient payload release[4].

Stenton et al. have described a thioether-containing linker that, upon exposure to Pd (0), releases an amine-linked payload (Figure 5 (A))[9]. Rossin et al. have recently developed an ADC bearing an antibody fragment and a trans-cyclooctene linker that, upon inverse-electron demand Diels–Alder (IEDDA) reaction with an externally administered tetrazine, releases an amine-containing payload in vivo (Figure 5 (B))[10].

The strategy of payload release based on photo-responsive cleavable triggers has gradually emerged in recent years. Photo-responsive cleavable triggers have the following advantages: (1) controlled drug release and effective reduction in off-target toxicity and (2) determination of the cleavage mechanism and no limitation on the intracellular release. Photo- responsive cleavable triggers enjoy the following advantages, including low toxicity, a rapid response, and high sensitivity and specificity[4].

In 2015, Nani et al. [11] firstly employed a near-infrared (NIR) light photo-caging strategy for ADCs (Figure 6 (A)). More recently, our group initially reported a novel ultraviolet (UV) light-controlled ADC (Figure 6 (B)) [12]. The linkers introduced a UV light-controlled O-nitrobenzyl group as a chemical trigger.

2 Enzyme Cleavable Linkers

In the classical mechanism of action, ADCs are trafficked to the lysosome of a cell where high concentrations of unique hydrolytic enzymes reside, thereby offering an opportunity for enzyme cleavable linkers to be selectively cleaved intracellularly. For selective payload release in the extracellular tumor area, enzymes that are upregulated in tumor microenvironments must be exploited[3].

Clip Chemistry: Diverse (Bio)(macro)molecular and Material Function through Breaking Covalent Bonds

YUAN Dingdong, 27 Oct 2022

The definition of click reaction is “The [click] reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation.” So, the author borrows this definition and gives the definition of clip reaction “Covalent bond breaking reaction that is modular and wide in scope. It must give very high yields and generate inoffensive byproducts, ideally in a stereo- and regiospecific manner. The process should involve simple reaction conditions and readily available starting materials and reagents.” Here, they give some examples of clip reactions in biomolecular application.

The first type is the Stoichiometric Clip Reaction; stoichiometric clip reactions require the stoichiometric addition of another reagent to trigger bond cleavage. For example, the disulfide reduction reaction. The process is often carried out in the laboratory using a phosphine-reducing agent, as shown in Figure 2a, and in chemical biology, we always used DTT or TCEP as the reductive reagents to reduce their disulfide bond in proteins to break their structure, such as the antibodies, also we use this method to free the cysteine for the next reactions. Also, the silyl ether−fluoride cleavage is another powerful clip reaction, which is most notably known for its role in the protection/ deprotection of alcohols; this method is widely used in organic synthesis; nowadays, this method is also used as the protect part for some biological application such as the drug release (Figure 2b). While the inverse electron demand Diels−Alder reaction of tetrazines and alkenes has been studied since at least the late 1950s, utilized in elegant total syntheses, and more recently popularized as an exceptional click reaction, its development as a clip reaction was only recently reported by Robillard and coworkers. Here, the clip reaction is triggered by a click reaction using modified tetrazines and trans-cyclooctenes and is driven by both dinitrogen extrusion (immediately following the cycloaddition step) and generation of aromaticity (formation of the 1,2-diazine). While these reagents are highly reactive when combined, they are quite stable on their own, giving this system many of the best characteristics of click and clip reactions (Figure 2a and 2c).

Hydrogel in medical biotechnology

HAN Yongxu, 29 Sep 2022

Hydrogels are typically defined as interconnected 3D molecular networks containing a high density of functional groups that enable the absorption and retention of large amounts of water. A wide variety of materials can be used to produce hydrogel networks. These materials can be broadly classified as having either synthetic or naturally occurring components. Synthetic building blocks, including polyacrylic acid, polyethylene glycol and so on. Meanwhile, natural components, including alginate, chitosan, and nucleic acids, in addition to proteins such as collagen, keratin and gelatin that naturally form hydrogels often possess increased biocompatibility and biological functionality when compared to synthetic hydrogels.

Hydrogel in biomedical exploration like drug delivery, tissue engineering, cell delivery, building scaffolds has become increasingly studied. For example, like Figure 2, the author reported a family of synthetic hydrogels that promote extensive organoid morphogenesis through dynamic rearrangements mediated by reversible hydrogen bonding. These tunable matrices are stress relaxing and thus promote efficient crypt budding in intestinal stem-cell epithelia through increased symmetry breaking and Paneth cell formation dependent on yes-associated protein. As such, these well-defined gels provide promising versatile matrices for fostering elaborate in vitro morphogenesis.

Another report shown in Figure 3, demonstrated that silver nanoparticle (AgNP)-based hydrogel enhances immunotherapy via the modulation of oral microbiota. The authors found a genus of bacteria (Peptostreptococcus) that could activate the immune system and improve the prognosis of patients. They had shown that in mice with subcutaneous or orthotopic murine OSCC tumours, combination therapy with the two components (nanoparticle-incorporating hydrogel and exogenous P. anaerobius) synergized with checkpoint inhibition with programmed death-1.

A Brief introduction to electron microscope

JIANG Hao, 19 May 2022

1. The limit of traditional optical microscope

Traditionally, scientists use optical microscope to observe microstructure of biological tissue and cells. But optical microscope have a limit in resolution. The limitation is caused by Rayleigh Criterion. The angular resolution of an optical system can be estimated (from the diameter of the aperture and the wavelength of the light) by the Rayleigh criterion defined by Lord Rayleigh: two point sources are regarded as just resolved when the principal diffraction maximum (center) of the Airy disk of one image coincides with the first minimum of the Airy disk of the other, as shown in Figure. 1.

2. Transmission electron microscope

To reach higher resolution, scientists turn to use electrons to observe the micro world. 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical microscope. An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. Electron microscopes use shaped magnetic fields to form electron optical lens systems that are analogous to the glass lenses of an optical light microscope.

The original form of the electron microscope, the transmission electron microscope (TEM), uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the “image”) may be viewed by projecting the magnified electron image onto a fluorescent viewing screen, a photographic film or the sensor of a digital camera.

Sulfonamide antibiotics and cephalosporins

ZHANG Yu, 12 May 2022

1. Sulfonamide antibiotics

Sulfonamide drugs were the first broadly used antibiotics and paved the way for the antibiotic revolution in medicine. Sulfonamide is a functional group act as competitive inhibitors of the enzyme dihydropteroate synthase (DHPS) which is an enzyme involved in folate synthesis. Folate is vital to the growth and development of the livings. Compared with the bacteria, human can acquire folate though the diet. Therefore, cutting of the folate supply by using the sulfonamide antibiotics can help us inhibit bacterial infection specifically without hurting ourselves. However, as a competitive inhibitor, the sulfonamide antibiotics can only inhibit the growth and multiplication of bacteria, but not kill them.^[1]

Prontosil, as Bayer named the new drug, was the first medicine ever discovered that could effectively treat a range of bacterial infections inside the body. It had a strong protective action against infections caused by streptococci, including blood infections, childbed fever, and erysipelas, and a lesser effect on infections caused by other cocci. However, it had no effect at all in the test tube, exerting its antibacterial action only in live animals. Later, Daniel Bovet, Federico Nitti, and Jacques and Thérèse Tréfouël found that the drug was metabolized into two parts inside the body, releasing a smaller, colorless, active compound called sulfonamide. ^[3]

The discovery of Prontosil paved the road for the development of sulfonamide antibiotics and filled the blank of the bacteria infection treatment before Penicillin. With the discovery of Penicillin, the sulfonamide antibiotics slowly faded out from the antibiotic market because of more side effects. However, sulfonamide drugs still work as diuretics or COX-2 inhibitors in pharmaceutical field.

GEFs play an important role in the control of small G proteins

QIU Jiaming, 5 May 2022

1. Rho superfamily

Rho GTPases are molecular switches that control a wide variety of signal transduction pathways inside cells. The Ras superfamily consists of more than 150 family members and can be divided into five categories, including five conserved subfamilies – Ras, Rho, Ran, Rab, and Arf(see Figure 1) – each with its own characteristics in terms of sequence, structural similarity, and cellular function. The Ras subfamily contains 36 members responsible for regulating signaling pathways involved in cell proliferation and for regulating signaling pathways involved in cell proliferation, morphology, differentiation, and cellular functions. The largest subfamily of the Ras superfamily is Rab, which consists of more than 60 members involved in vesicle and protein transport in cells. 30 members of the Arf family act as regulators of intracellular transport. The Ras subfamily consists of 36 members and is responsible for regulating signaling pathways involved in cell proliferation. The 20 members of the Rho subfamily are responsible for the regulation of signaling pathways involved in cell proliferation, transport and differentiation, and cell survival. The 20 members of the Rho subfamily are key regulators of actin organization, gene expression and cell cycle progression. The largest one subfamily, Rab, consists of more than 60 members and is involved in vesicle and protein transport. involved in vesicle and protein transport. There are 30 members of the Arf protein members that are also regulators of intracellular transport.Ran subfamily. The Ran subfamily has only one protein, which is the most abundant small GTPase in the cell. It is the most abundant small GTPase in the cell and is involved in nuclear transport.

2. GEFs and GAPs

Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate the activity of small guanine nucleotide-binding (G) proteins for the purpose of regulating cellular functions (See Figure2). In general, GEFs regulate signaling by catalyzing the exchange of G protein-bound GDP to GTP, whereas GAPs terminate signaling by inducing GTP hydrolysis. Recent evidence suggests that these proteins may be potential therapeutic targets for the development of drugs to treat a variety of diseases, including anticancer antibacterial. GEFs contain a variety of species, all of which activate Cdc42 by mediating the exchange of GDP with GTP in Cdc42. during activation, when receiving external signals, GEFs such as Dbl bind to inactive Cdc42, forming a Cdc42-GEF binary complex, causing GDP binding to fall off from small G proteins, followed by the release of GTP from Cdc42 due to the cell membrane when GTP can be more than ten times that of GDP. GEF binds with low affinity to active Cdc42 and is therefore released from Cdc42-GTP. Active Cdc42 interacts with downstream effectors such as N-WASP to produce biological functions such as actin polymerization. GTPase activating proteins or GAPs promote GTP hydrolysis of Cdc42 and return Cdc42 to its basal inactive state

Emerging New Concepts of Degrader Technologies

YUAN Dingdong, 28 April 2022

Human genetic studies have shown that many novel target proteins cause disease by acquiring functional toxicity. Traditional drug development strategies need to occupy binding sites to inhibit the functional activity of target proteins, which makes disease-related scaffold proteins, transcription factors, and other non-enzymatic proteins “undruggable”. These “undruggable” disease-causing proteins can be corrected or degraded by enhancing protein quality control systems.

Selective degradation of pathogenic protein levels is a more effective strategy. Nucleic acid-based RNA- or DNA-targeting agents for gene silencing are one approach, while gene therapy is promising, it still faces some key challenges such as being difficult to deliver and prohibitively expensive. Therefore, inducing degradation of target proteins is more feasible and can meet clinical needs (Fig. 1).¹

• 2. Total synthesis of quinine

Small molecules are able to induce selective degradation of the protein of interest (POI) by adding tags recognized by the degradation machinery. In 2011-2012, Crews and colleagues reported POI degradation induced by small molecule hydrophobic tags (HyT). HyT is achieved by a molecule composed of a hydrophobic segment and a ligand that binds to POI. Thus, HyT can add hydrophobic fragments to the POI, resulting in structural changes of the target protein and subsequent proteolysis (Fig. 1).¹

PROTAC

At present, the main method of small molecule-induced protein degradation is the protein degradation targeted chimera (PROTAC) technology. The PROTAC molecule increases the ubiquitination efficiency of the target protein by shortening the distance between the target protein and E3 ligase, thereby inducing the proteasome system to degrade the target protein. However, PROTAC has certain limitations. For example, this method relies on a specific E3 ligase, which limits its application in certain cells. Its molecular weight is generally too large, and it cannot target the secret proteins and membrane proteins (Fig. 1).¹

LYTAC

LYTAC (Lysosome Targeted Chimera) technology is a method that utilizes the endosome/lysosome pathway to degrade target proteins. PROTAC technology mainly targets intracellular proteins, while LYTAC can act on membrane proteins and extracellular proteins that are resistant to PROTAC (such as EGFR). LYTAC molecules consist of antibodies against a specific target protein and are covalently linked to mannose 6-phosphate (M6P). Antibodies are recognized by endogenous systems, and their bound target proteins are transported to lysosomes for degradation. The advantage of LYTAC technology is that it utilizes the endogenous degradation pathway to degrade extracellular proteins and membrane proteins. The main disadvantage is that the molecular weight is large, and the antibody or polypeptide in the molecule may induce an immune response (Fig. 2).²

In March 2021, they published a new work in the journal Nature Chemical Biology, revealing a LYTAC technology that utilizes the asialoglycoprotein receptor (ASGPR). Since ASGPR is a liver-specific lysosomal targeting receptor, LYTAC technology utilizing ASGPR is able to degrade extracellular proteins in a cell-type-restricted manner. Specifically, the study conjugated the binder triantenerrary N-acetylgalactosamine (tri-GalNAc) of ASGPR to the target protein through a linker. Based on these findings, the researchers believe that GalNAc-LYTAC represents a cell-type-restricted lysosomal protein degradation pathway that further paves the way for the clinical translation of LYTAC technology (Fig. 2).³

Story of Quinine: Structure, History, and Total Synthesis

BAO Yishu, 21 April 2022

1. Discovery of quinine

Tonic water glows faintly in sunlight and this phenomenon is even more apparent under ultraviolet light irradiation. It arises from tonic water’s main ingredient, quinine. The glow of a quinine solution was firstly noticed by the polymath Sir John Herschel.¹ A few years later, this phenomenon was called “fluorescence” by the physicist and mathematician Sir George Stokes.

Quinine is an alkaloid extracted from the bark of Cinchona trees indigenous to South American forests, which attracted intense interest worldwide on account of its prophylactic characteristics. Its name is derived from quina-quina and it can be roughly translated as bark of barks. Native South Americans used it to alleviate shivering, fever, and pain.² Later, they found that quinine also promptly relieved the symptoms of malaria and quinine became well-known around the world for it. Before artemisinin was discovered, quinine was the drug of choice for the treatment of malaria (Figure 1).

• 2. Total synthesis of quinine

The chemical structure of quinine is complex and its total synthesis confounded chemists for over a century since its first isolation from the bark in 1820 by two French pharmacists. In 1856, Sir William Henry Perkin who was only 18 years old, attempted to synthesize quinine by oxidizing N-allyltoluidine. He failed but he generated mauveine which is the first synthetic organic dye to be synthesized on a large scale for industrial and commercial purposes (Figure 2).³ Subsequent attempts by Paul Rabe and Karl Kindler in the early twentieth century. This sequence involved: (1) oxidation of d-quinotoxine with sodium hypobromite to produce N-bromoquinotoxine; (2) base-mediated cyclization to produce quininone; and (3) aluminum powder reduction of quininone to produce quinine and quinidine (Figure 3).⁴

Regarding the breakthrough progress in the total synthesis of quinine, we must mention an organic chemistry legend, Robert Burns Woodward. Woodward was born in Boston in 1917. In 1933, 16-years-old Woodward enrolled as an undergraduate at Massachusetts Institute of Technology (MIT). In 1936, he received a Bachelor of Science degree. Only one year later, MIT awarded him a doctorate. In 1944, World War II broke out, which made the spread of malaria especially fast, resulting in a short supply of the quinine. At this time, Woodward made an amazing decision to totally synthesize quinine. He and his postdoctoral researcher William von Eggers Doering reported the synthesis of quinine, starting from using 3-hydroxybenzaldehyde, through 55 reactions including condensation, cyclization, alkylation, reduction, acetylation, oxidation, and so on. The final step is the Claisen condensation and finally, the generation of the racemic quinidone after acid treatment (Figure 4).⁵

In addition to quinine, Woodward and his research group’s achievements in total synthesis are breathtaking: cholesterol, cortisone, lanosterol, strychnine, tetracylines, colchicine, reserpine, lysergic acid and ergonovine, chlorophyll, cephalosporin C, erythromycin, and vitamin B12 (the last, with Albert Eschenmoser). Those are just a few among the herd of his synthetic accomplishments. In 1965, Woodward won the Nobel Prize in Chemistry for his outstanding achievements in organic synthesis.

Photobiocatalysis-a new way for transformation

CHEN Hongfei, 14 April 2022

1. The era of antibiotic discovery

Biocatalysis reaction especially enzyme reaction has advantages in high chemical selectivity and good stereoselectivity. However, this kind of reaction always has limited substrates scope. On the other hand, photocatalysis reaction which using visible light as energy source can generate open-shell intermediates for different reactions. But the poor stereoselectivity is one of the drawbacks of this kind of reaction. Recent years, researchers combine the biocatalysis and photocatalysis together for new transformations which based on visible light and enzymes. The photobiocatalysis are divided into three main parts: a) natural photoenzymes; b) new reactivity of cofactor dependent enzymes; c) synergistic combination of photocatalyst and enzymes (Fig.1.).^[1]

The first kind of photobiocatalysis is natural photoenzyme. A recent example is the discovery and characterization of a fatty acid photodecarboxylase (CvFAP). CvFAP was first reported by Beisson and co-workers in 2017. This enzyme can use FAD and light to produce alkanes and alkenes from long-chain fatty acids (Fig.2.). ^[2,3]

Since the discovery of the newly characterized CvFAP, a variety of photoenzymatic transformations have been disclosed by tuning substrate specificity and function. Very recent studies of engineering CvFAP for a photodecarboxylative deuteration and for a selective photodecarboxylation of trans fatty acids were reported by Wu and co-workers. Given the growing number of applications using this newly discovered CvFAP, it is sure that the discovery of other new photoenzymes will enable the development of new to- nature chemical transformations.^[4]

The second kind of photobiocatalysis is new reactivity of cofactor dependent enzymes. The redox active cofactors of these enzymes can form electron donor−acceptor (EDA) complexes with unnatural substrates which can generate radical species under mild conditions through single-electron transfer (SET) events. In 2016, Hyster and co-workers demonstrated the first example of the use of photoexcited enzyme−substrate complexes for achieving non-natural reactivity in enzymes. They discovered NADPH-dependent ketoreductases, which normally catalyze the nonlight-driven reduction of carbonyl functional groups, could catalyze the radical dehalogenation of halolactones through the photoexcitation of a halolactone-NADPH EDA complex. Very recently, they expanded the scope of the reductive cyclization to chiral tertiary amine synthesis by altering the radical acceptor from a tethered C=C double bond to an oxime (Fig.3.). ^[5,6,7]

The third kind of photobiocatalysis is synergistic combination of photocatalyst and enzymes. Currently, there are three strategies to synergize external photoredox catalysts and enzymes for abiological transformations. In each strategy, the enzyme is used to control selectivity of the reaction, and the external photoredox catalyst can be used for (I) selective single-electron reduction of the enzyme-bound substrate, (II) substrate racemization enabled by the photoinduced SET, or (III) E/Z-isomerization of alkenes via energy transfer. In the last five years, researchers have leveraged the reactivity of photocatalysts and selectivity of enzymes for difficult asymmetric syntheses. It is expected that emerging strategies for dual photocatalysis, and enzyme catalysis can be applied to enzymes without light absorbing cofactors and that the compatibility of photocatalysts with enzyme cofactors could be further explored for abiological transformations. ^[8,9,10]

Computer-aided engineering of enzymatic selectivity

WANG Yue, 7 April 2022

Life has evolved an astonishing array of enzymes that play a wide range of roles in vital biological processes. Intriguingly, enzymes with such remarkable power, selectivity, and sustainability strongly encourage biochemists to supplement or even replace some traditional chemical synthesis routes on a large scale. Although the trend toward biosynthesis of natural products or blockbuster drugs in diverse industries strongly drives the development of enzymes that are capable of catalyzing challenging reactions, even non-natural ones, there is still a mismatch between the inefficiency of enzymes toward non-native substrates and the need for complex chemicals in diverse industries.

The development of credible mechanistic insights requires a high-quality structure in knowledge-based protein engineering. Compared to decades ago, enzyme crystallization has advanced significantly, but is still a much slower process than sequence accumulation. As a result, a variety of computer-based prediction approaches are available that can effectively provide acceptable enzyme structures, which are mainly homology alignments based on templates and conformational sampling strategies without templates (Kuhlman and Bradley, 2019). Meanwhile, machine learning has also been used to explore deeper buried information in structure databases to improve the accuracy of structure prediction, especially in the case of the latest alphafold2 in CASP14 (Service, 2020).

By interacting with the substrate, enzymes perform their catalytic function. As a fundamental tool to predict the optimal binding mode of ligands and enzymes, molecular docking was inspired by “lock-and-key” (Fischer, 1894), “induced-fit” (Koshland, 1958), and “configurational selection” (Kumar et al., 2000) models. These approaches included rigid docking, semi-flexible docking, flexible docking, and the latest CaverDock, which considered substance transport in enzyme channels. The precondition for excellent molecular docking results is the use of high-quality structures, which can be obtained either experimentally or computationally.

Static snapshot structures are often inadequate to analyze dynamic enzyme-catalyzed processes, including substrate binding, chemical bond cleavage, and product release. Dynamic conformational ensembles are continuously sampled using MD simulation, a powerful computational scheme based on classical mechanics (Hollingsworth and Dror, 2018). Additionally, MD simulations of large biosystems are approaching experimentally relevant timescales, raising the possibility of mutually reinforcing experimental and theoretical findings, providing new opportunities in rationalizing and engineering enzymes (Cerutti and Case, 2019; Franz et al., 2020; Huggins et al., 2019; Surpeta et al., 2020). At present, various useful MD methods have been developed, such as MD simulations incorporating enhanced sampling to reveal ligand migration (Rydzewski and Nowak, 2017) and coarse-grained MD simulations to cope with multiscale biological processes (Kmiecik et al., 2016; Souza et al., 2020).

Antibiotic resistance crisis-the sword of Damocles hanging in the human head

Chunjian Wu, 24 March 2022

1. The era of antibiotic discovery

For most of human history, bacterial pathogens have been a major cause of disease and mortality. In thepre-antibiotic era of the early 1900s, people had no medicines against these common germs and as a result, human suffering was enormous. Death from infection could follow from something as minor as a scratch. But this situation changes after the first antibiotic—penicillin was discovered in 1928 by Scottish scientist Alexander Fleming. Suddenly, the morbidity and mortality sharply were reduced by this“miracle drugs”, which also raises a lot of attention all over the world. Shortly afterward, the golden era of antibiotic launched, and the main classes of antibiotics were discovered in a fairly short period of time (Figure 1).

Antibiotics have revolutionized the field of medicine. They have saved millions of lives each year, alleviated pain and suffering, and have even been used prophylactically for the prevention of infectious diseases. What is more, antibiotics have been crucial in the increase in life expectancy in the United States from 47 years in 1900 to 74 years for males and to 80 years for females in the year 2000 (The Journal of antibiotics, 70(5), pp.520-526.). But the discovery of antibiotic resistance appeared as a disturbing storm-cloud in this clear sky.

The crisis of antibiotic resistance

In fact, such a continuous progress in discovery of new antibiotics in the 1940–1980 period was mostly fostered by the necessity of “fighting new antibiotic resistances,” where the new discoveries compensated for the emergence of new resistances in bacterial pathogens. The golden era ended rather abruptly in the early 1960s (Figure 1). Infectious disease is now the second leading killer in the world, number three in developed nations and fourth in the United States (The Journal of antibiotics, 70(5), pp.520-526.). For example, current worldwide deaths attributable to antimicrobial resistance have been estimated at ~700,000 per year, rising to 10 million per year by 2050 if present trends continue (Science, 2021, 373(6554), pp.471-471.).

Despite the fact that the spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide, pharmaceutical research and development has failed to meet the clinical need for new antibiotics. In the past 20 years, only two new antibiotic classes (lipopeptides and oxazolidinones) have been developed and approved by international drug agencies (US Food and Drug Administration and European Medicines Agency)—both of which provide coverage against Gram-positive bacteria. More sadly, the drugs against Gram-negative bacteria have not been developed after the last novel drug, quinolones , discovered in 1962

The World Health Organization (WHO) recently introduced a list of priority pathogens (The Lancet Infectious Diseases, 18(3), pp.318-327.) and noted those of “critical priority”-drug-resistant Enterobacteriaceae (E. coli, Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter), Pseudomonas aeruginosa, and Acinetobacter baumannii, which are responsible for a global health problem. All of the critical priority pathogens are Gram-negative bacteria. The antibiotic resistance crisis threatens the return of the pre-antibiotic era of epidemics and pandemics. It is important to note that antibiotics not only save lives, but enable modern medicine. Without antibiotics, surgery, chemotherapy, or organ transplantation become highly problematic.

Targets of antibiotics targets and resistance mechanisms

Antibiotics are available that effectively inhibit bacterial cell wall synthesis, protein synthesis, and DNA replication (Figure 3a). But bacteria have evolved an array of mechanisms that enable them to resist the inhibitory action of antibiotics. There exists by now an extensive body of knowledge regarding the nature of different mechanistic classes of antibiotic resistance (Figure 3a). Most such mechanisms serve either to reduce antibiotic concentration in the vicinity of the molecular target (through active efflux or chemical inactivation of the antibiotic) or involve a fundamental change in the nature of the target that reduces the antibiotic’s inhibitory effect (for example, as a consequence of mutation or chemical modification). Recent work has revealed that target protection is a key mechanistic player in clinically significant antibiotic resistance that affects diverse classes of antibacterial drugs and is prevalent in bacterial pathogens.

Production of Semi-synthetic Artemisinin in engineered yeast

Jiang Hao, 17 March 202

1. Artimisinin, a key in the battle against malaria

In 2016, malaria killed an estimated 445,000 people, 90% of them in Africa. That is, one person dies every 71 seconds, caused by preventable and treatable diseases. Despite a 25% drop in deaths since 2010, the World Health Organization recently warned that progress was stalling and called for a redoubled effort to combat the scourge.

Some of the most critical weapons in this battle are artemisinin-based combination therapy (ACT), considered the most effective antimalarial drug. They contain derivatives of artemisinin, a molecule found in the sweet wormwood (Artemisia annua) plant, which grows mainly in China and Vietnam.

But the world supply of artemisinin has been very unstable over the past 15 years. As more farmers grow artemisinin to meet growing demand, the supply of artemisinin has increased and pushed prices down. So, farmers turned to more profitable crops. The supply of artemisinin then went down, and prices soared again. The artemisinin supply roller coaster has hindered efforts to make ACTs available to everyone who needs them.

There are various methods of introducing foreign nucleic acids into a eukaryotic cell: some rely on physical treatment; others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological [1].

2. Commericial production of semi-synthetic artemisinin

Alternative sources of artemisinin can solve this problem. Back in 2004, the Bill & Melinda Gates Foundation funded a project to develop a genetically engineered yeast that could make artemisinic acid, a compound just steps away from artemisinin itself. Sanofi eventually commercialized the process in 2013 to produce semi-synthetic artemisinin (SSA), capable of meeting about one-third of global demand.

To reach this achievement, we must fully understand the biosynthetic pathway of artemisinin. It is mainly divided into four parts:

Formation of farnesyl pyrophosphate FPP through both mevalonate pathway (MVA) and non-mevalonate pathway (DXP).

Under the action of amorpha-4,11-diene synthase, FPP is cyclized to form amorphine-4,11-diene, the intermediate of artemisinin.

Under the action of amorpha-4,11-diene oxidase, amorpha-4,11-diene is further oxidized to form artemisinol and artemisinaldehyde, and then artemisinic acid are synthesized.

Chemical-based Transfection and Its Applications in Vaccines

Li Biquan, 10 March 2022

1. Introduction to Transfection

Transfection, which is the process of introducing foreign nucleic acids into host cells, is one of the most powerful molecular biology tools currently in use. In animal cells, transfection is the preferred term. Additionally, we should pay attention to the differences of the concept between transfection, transformation, and transduction.

Generally, we can divide transfection into categories, stable and transient transfection, which differ in their long-term effects on a cell. A stably-transfected cell will continuously express transfected DNA and pass it on to daughter cells, while a transiently-transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells. From Figure 1, we could compare the differences between stable and transient transfection, and we can note that in the process of stable transfection, the DNA would be integrated into the gene of the host cell, while transient transfection doesn’t experience this step [1].

There are various methods of introducing foreign nucleic acids into a eukaryotic cell: some rely on physical treatment; others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological [1].

2. Chemical-based Transfection

In this abstract, we are mainly to introduce chemical-based transfection. As shown in Figure 2, Chemical-based transfection can be divided into several kinds: calcium phosphate, cationic polymers, liposomes (lipofection) and, nanoparticles etc [1].

One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973 [2]. This process has been a preferred method of identifying many oncogenes and proved that the complex access to the cell through endocytosis. However, the transfection efficiency strongly depends on the cell constitution, the pH, and the quality and the amount of the used nucleotides, and it is harmful and therefore not suitable for most sensitive primary cell lines [1].

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. This method can be transiently transfected into eukaryotic cells in an inexpensive and simple manner and is able to be easily reproduced, but the efficiency in a number of cell types is low (including primary cells) and it is cytotoxic and therefore not suitable for sensitive cells [3].

Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer [4]. Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material [5]. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, PEI and, calcium phosphate [6]. This method is easy to apply and yields highly reproducible results of transient and stable transfections, but the transfection efficiency strongly depends on the cell type. Another disadvantage is that he viability after the transfection process might be decreased due to the high cellular sensitivity, especially for primary and non-dividing cells. It is worth noting that lipofection is based on endosomal molecule uptake, and also can easily merge with the cell membrane since they are both made of a phospholipid bilayer [5].

The last method we’d like to introduce and use in vaccines is nanoparticle-based transfection. Some cells are relatively easy to transfect, whereas others, such as stem cells, suspension cells, or primary cells show poor transfection results. This is where nanoparticles, each behaving as a single unit, come into the play, owing to their ultra-miniaturized size (ranging from 1 to 100 nm) [1, 7]. This method is used in many examples, such as AuNPs-based CRISPR delivery [8] and LNPs-based mRNA (vaccine) delivery [9, 10], which is shown in Figure 3.

LNP-based delivery is one of the most and main ways in mRNA vaccines delivery, other major delivery methods for mRNA vaccines Commonly used delivery methods and carrier molecules for mRNA vaccines are shown in Figure 4. [11]

Proximity labeling: a new tool to explore molecular interactions

Zhang Yu, 17 Feb. 2022

Many biological processes are regulated through the molecular interactions of proteins and nucleic acids.  In the past several years, affinity purification and yeast two-hybrid have been widely applied to discover potential molecular interactions. (1,2) Antibody-based affinity purification, in combination with mass spectrometry–based proteomics, allows the enrichment and identification of stable interaction partners of specific proteins of interest. The main limitation of affinity purification, however, is that weak or transient interactions are often lost during cell lysis and the subsequent washing steps. Moreover, affinity purification is challenging to apply to insoluble targets or protein baits.

Yeast two-hybrid and other protein complementation assays represent another approach for mapping protein–protein, protein–RNA and protein-DNA interactions in living cells. (1) These approaches are often high throughput, enabling the screening of thousands to millions of potential molecular interactions. (3) However, many proteins complementation assays have cell type and organelle type restrictions.

Then, with the development of the molecular biology, a brand-new technology was developed—the proximity labelling. Proximity labeling (PL) is a technology used to find the interaction partners of ‘baits’ proteins by fusing ‘baits’ proteins with enzymes who can modified proximity molecules. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. (4) The predecessor of the proximity labelling is DamID. It is a method developed in 2000 by Steven Henikoff for identifying parts of the genome proximal to a chromatin protein of interest. (5) DamID relies on a DNA methyltransferase fusion to the chromatin protein to nonnaturally methylate DNA, which can then be subsequently sequenced to reveal genome methylation sites near the protein. Researchers were guided by the fusion protein strategy of DamID to create a method for site-specific labeling of protein targets, culminating in the creation of the biotin protein labelling-based BioID in 2012. (6) Alice Ting and the Ting lab at Stanford University have engineered several proteins that demonstrate improvements in biotin-based proximity labeling efficacy and speed.

Potential of AlphaFold in Determining Protein Structure

Han Yongxu, 10 Feb. 2022

Centra dogma uncovers the production of protein which was translated from mRNA caring the genetic information of DNA. Protein-related research is the most fertile areas in chemical biology and medical biology. Function of protein is determined by its conformation which is complicated and variable, thus deciphering how protein worked entails the illustration of the structure.

The determination of protein structures can be dated from 60 years ago. John Kendrew, as a biologist from England, was awarded the Nobel Prize in chemistry in 1962 due to the contribution on the determination of myoglobin which containing heme with x-ray crystallography. Several scientist were also granted the Nobel Prize for the discovery on the conformation of proteins like virus particles, rhodopsin protein, aquaporins and ion channel protein and so forth in the following years. X-ray diffraction crystallography, NMR and cryo-EM were major methods to explore the protein structures. Searching from the Protein Data Bank (PDB), we can found that there are about 180,000 released entries for protein structures, among of them, more than 80% were determined from X-ray crystallography, 13,448 were solved by NMR and no more than 10, 000 were from cryo-EM (1). X-ray diffraction crystallography was one of the first experimental methods used for structural resolution, however, X-ray diffraction cannot be applied to larger proteins (2). NMR was generally believed to be able to describe the real structure of proteins in solutions rather than crystal structure. Moreover, NMR analysis can obtain the structural position of hydrogen atom. However, due to the unstable structures of proteins in solution sometimes, it is difficult to capture stable signals, thus computer modeling and other methods are supplemented for the structure determination (3). Cryo-EM method in resolving bound ligands and delineating structured water molecules makes it possible to obtain detailed information relevant to molecular mechanisms (4).

AlphaFold known from Critical Assessment of Structure Prediction (CASP) conference is a neural-network-based approach to predicting protein structures with high accuracy. Recently, the source code and almost 350,000 protein models within AlphaFold 2 from various species, including human and bacterial have been made public (5).  

The network of AlphaFold can directly predict 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs, then apply multiple sequence alignments (MSAs) and pairwise features to illustrate a new architecture. There are two index including the predicted local-distance difference test (pLDDT) and template modelling score (TM-score) to judge the prediction efficiency. pLDDT can reliably predict the Cα local-distance difference test (lDDT-Cα) accuracy of the corresponding prediction, and the latter one presents the global superposition metric template modelling score (6).

Protein Liquid-Liquid Phase separation

Liu Min, 20 Jan. 2022

Since 2009, when Hyman’s group discovered the liquid property of P granules from C. elegans, most of the research hotspots focused on the protein condensate. Actually, since 2000, many scientists have discovered various membrane-less structures. Most of these structures are 100-1000 nm diameters and contain a large amount of protein and RNA, which are involved in the normal physiological activities of cells. For example, stress granules are structures associated with translation regulation, while dvl2 puncta are associated with cell signaling.

Liquid-liquid separation is a very common phenomenon in the field of physical chemistry. Many substances are single phase in the monomer stage, and when polymerized they form two phases. The protein phase separation is more complicated than polymer, and the two most important driving forces are the disorder of the protein sequence and the multivalent effect. For example, simply increasing the concentration of a protein component can see phase separation, increasing the valency of a component can also promote phase separation, or reducing the solubility of a component can also help phase separation.

In 2012, Rosen’s group from Southwestern Medical Center reconstituted the phase-separated system for the first time in vitro. They successfully realized the observation of phase-separated droplets by increasing the valency of the two interacting proteins. Afterwards, Professor Clifford P. Brangwynne from Princeton did a lot of interesting work on the phase separation system from discovery, establishment to application, which are related to the transcription and translation of genes.

The virulence mechanisms of EPEC

Qiu Jiaming, 13 Jan. 2022

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen, which can cause significant paediatric morbidity and mortality all over the world, especially in developing countries. The infection of EPEC involves four stages: attachment, sending in effector proteins, actin reorganization, and pedestal formation and includes various elements which play a vital role in EPEC infection.

T3SS

Firstly, EPEC uses locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and various cognate effectors (Tir, Map, EspF, EspG, EspH, and EspI) to infect host cells. Through the integration of coordinated intracellular and extracellular cues, the T3SS is assembled within the bacterial cell wall, as well as the plasma membrane of the host cell. T3SS construction includes four stages: (i) assembly of the basal body and export apparatus, (ii) assembly of the inner rod and needle, (iii) assembly of the filament and translocon, and (iv) secretion of effectors. And along with the formation of injectisome, EPECs can complete the infection.

Intimin and Tir

Secondly, the especially feature of A/E bacterial pathogens is the formation of pedestal beneath tightly adherent bacteria and localized disruption of the brush border of the intestine. And in this process, the intimin which belongs to bacterial outer membrane adhesin, is required for the production of A/E lesions and diarrhea. As we known, EPEC use T3SS to translocate their own intimin receptor, Tir, to the membranes of mammalian cells. The translocated Tir triggers additional host signaling events and actin nucleation. Actin nucleation is critical for lesion formation. Current studies have demonstrated that when the IBD domain of Tir binds to the D3 domain of the EPEC intimin protein on the mammalian cell surface, the bacteria are primed to remain on the mammalian cell surface.

Bioorthogonal reactions

Yuan Dingdong, 22 Dec. 2021

Bioorthogonal chemistry means any chemical reaction that may occur inside living systems without interfering with native biochemical processes. This concept was coined by Carolyn R. Bertozzi in 2003^[1]. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody–drug conjugation and drug delivery. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide–alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions (Fig. 2) ^[2].

Amongst all bioorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazines (s-tetrazines, Tz) and various dienophiles, referred as inverse electron demand Diels–Alder (IEDDA) reaction, is the one that satisfies most of the bioorthogonal criteria (e.g., fast, selective, biocompatible and catalyst-free) necessary for use in applications from protein labelling to cancer imaging or materials science. Diels–Alder [4+2]-cycloaddition describes the reaction between a diene (e.g., 1,2,4,5-tetrazines) and a dienophile (alkene or alkyne) to form a six-membered ring in a π4s + π2s fashion, via suprafacial/suprafacial interaction of 4π-electrons of the diene with the 2π-electrons of the dienophile (Fig. 3) ^[3].

Tetrazine can react with rich and varied strained or unstrained dienophiles, such as trans-cyclooctene (TCO), cyclopropane, norbornene and styrene which can be tuned to reach rate constants from 1 up to 10⁶ M^-1s^-1 with different types of dienophiles (Fig. 4) ^[3].

Evolution of Directed Evolution Methodology

Liu Miao, 8 Dec. 2021

In 1859, Darwin’s On the Origin of Species founded one of the most important theory in biology, the theory of evolution. Although elution theory itself has experienced an evolutionary process, the basic concept of variation–selection–retention cycle unchanged. Adopted from the evolution theory, evolving proteins by mutagenesis and selection was proposed, and in 1993, Arnold and co-workers turned this concept into practice. Since then directed evolution has been widely used to obtain powerful biomolecules. The general procedure of directed evolution includes two main steps: gene diversification and screening/selection (Figure 1). The improved variant will serve as a new starting point for the next round of gene diversification to form a cycle. With the development of chemistry, biochemistry and molecular biology, strategies used in directed evolution have also been evolved.

1. Development of Gene Diversification

In molecular evolution, gene mutation and recombination are two major forces for diversification. For creating gene mutation, random mutagenesis by error-prone PCR was firstly used as the experiment is convenient to carry out, but high-throughput screening methods are necessary because of its random nature. With more knowledge of the protein structure–function relationships revealed by biochemist, focused mutagenesis like site saturation mutagenesis can be performed on critical residues of enzymes, thus creating a smaller but more efficient library. For gene recombination, various DNA shuffling methods were utilized as more molecular biology tools has been developed to imitate the natural process of homologous recombination.

Beside create mutants in test tube, which is labor and time intensive, in vivo gene diversification methods were also developed as more in vivo gene edit tools were found. Early mutation applications in microorganism use E. coli carrying with gene mutation enzyme plasmid, but their applications were limited since they introduce deleterious mutations in the host genome and result in genetic instability. So the development of in vivo gene mutation focused on finding orthogonal mutator plasmids and strains. For in vivo gene recombination, molecular biology tools like bacteriophage λ Red system and Cre-LoxP system were applied. In recent years, CRISPR-Cas system was proved to be a powerful gene edit tool in prokaryotic and eukaryotic cells, which was also used to introduce gene diversifications in vivo.