Molecular Modeling Methods in the Development of Affine and Specific Protein-Binding Agents.

High-affinity and specific agents are widely applied in various areas, including diagnostics, scientific research, and disease therapy (as drugs and drug delivery systems). It takes significant time to develop them. For this reason, development of high-affinity agents extensively utilizes computer methods at various stages for the analysis and modeling of these molecules. The review describes the main affinity and specific agents, such as monoclonal antibodies and their fragments, antibody mimetics, aptamers, and molecularly imprinted polymers. The methods of their obtaining as well as their main advantages and disadvantages are briefly described, with special attention focused on the molecular modeling methods used for their analysis and development.

AliNA - a deep learning program for RNA secondary structure prediction.

Nowadays there are numerous discovered natural RNA variations participating in different cellular processes and artificial RNA, e. g., aptamers, riboswitches. One of the required tasks in the investigation of their functions and mechanism of influence on cells and interaction with targets is the prediction of RNA secondary structures. The classic thermodynamic-based prediction algorithms do not consider the specificity of biological folding and deep learning methods that were designed to resolve this issue suffer from homology-based methods problems. Herein, we present a method for RNA secondary structure prediction based on deep learning - AliNA (ALIgned Nucleic Acids). Our method successfully predicts secondary structures for non-homologous to train-data RNA families thanks to usage of the data augmentation techniques. Augmentation extends existing datasets with easily-accessible simulated data. The proposed method shows a high quality of prediction across different benchmarks including pseudoknots. The method is available on GitHub for free (https://github.com/Arty40m/AliNA).

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Oxazolinyl derivatives of androst-16-ene as inhibitors of CYP17A1 activity and prostate carcinoma cells proliferation: Effects of substituents in oxazolinyl moiety.

Steroid derivatives modified with nitrogen containing heterocycles are known to inhibit activity of steroidogenic enzymes, decrease proliferation of cancer cells and attract attention as promising anticancer agents. Specifically, 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole 1a potently inhibited proliferation of prostate carcinoma cells. In this study we synthesized and investigated five new derivatives of 3ß-hydroxyandrosta-5,16-diene comprising 4'-methyl or 4'-phenyl substituted oxazolinyl cycle 1 (b-f). Docking of compounds 1 (a-f) to CYP17A1 active site revealed that the presence of substitutents at C4' atom in oxazoline cycle, as well as C4' atom configuration, significantly affect docking poses of compounds in the complexes with enzyme. Testing of compounds 1 (a-f) as CYP17A1 inhibitors revealed that the only compound 1a, comprising unsubstituted oxazolinyl moiety, demonstrated strong inhibitory activity, while other compounds 1 (b-f) were slightly active or non active. Compounds 1 (a-f) efficiently decreased growth and proliferation of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the effect of compound 1a was the most powerful. Compound 1a efficiently stimulated apoptosis and caused PC-3 cells death, that was demonstrated by a direct comparison of pro-apoptotic effects of compound 1a and abiraterone.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4.

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

New Positive TRPC6 Modulator Penetrates Blood-Brain Barrier, Eliminates Synaptic Deficiency and Restores Memory Deficit in 5xFAD Mice.

Synapse loss in the brain of Alzheimer's disease patients correlates with cognitive dysfunctions. Drugs that limit synaptic loss are promising pharmacological agents. The transient receptor potential cation channel, subfamily C, member 6 (TRPC6) regulates the formation of an excitatory synapse. Positive regulation of TRPC6 results in increased synapse formation and enhances learning and memory in animal models. The novel selective TRPC6 agonist, 3-(3-,4-Dihydro-6,7-dimethoxy-3,3-dimethyl-1-isoquinolinyl)-2H-1-benzopyran-2-one, has recently been identified. Here we present in silico, in vitro, ex vivo, pharmacokinetic and in vivo studies of this compound. We demonstrate that it binds to the extracellular agonist binding site of the human TRPC6, protects hippocampal mushroom spines from amyloid toxicity in vitro, efficiently recovers synaptic plasticity in 5xFAD brain slices, penetrates the blood-brain barrier and recovers cognitive deficits in 5xFAD mice. We suggest that C20 might be recognized as the novel TRPC6-selective drug suitable to treat synaptic deficiency in Alzheimer's disease-affected hippocampal neurons.

New Genetic Bomb Trigger: Design, Synthesis, Molecular Dynamics Simulation, and Biological Evaluation of Novel BIBR1532-Related Analogs Targeting Telomerase against Non-Small Cell Lung Cancer.

Telomeres serve a critical function in cell replication and proliferation at every stage of the cell cycle. Telomerase is a ribonucleoprotein, responsible for maintaining the telomere length and chromosomal integrity of frequently dividing cells. Although it is silenced in most human somatic cells, telomere restoration occurs in cancer cells because of telomerase activation or alternative telomere lengthening. The telomerase enzyme is a universal anticancer target that is expressed in 85-95% of cancers. BIBR1532 is a selective non-nucleoside potent telomerase inhibitor that acts by direct noncompetitive inhibition. Relying on its structural features, three different series were designed, and 30 novel compounds were synthesized and biologically evaluated as telomerase inhibitors using a telomeric repeat amplification protocol (TRAP) assay. Target compounds 29a, 36b, and 39b reported the greatest inhibitory effect on telomerase enzyme with IC50 values of 1.7, 0.3, and 2.0 µM, respectively, while BIBR1532 displayed IC50 = 0.2 µM. Compounds 29a, 36b, and 39b were subsequently tested using a living-cell TRAP assay and were able to penetrate the cell membrane and inhibit telomerase inside living cancer cells. Compound 36b was tested for cytotoxicity against 60 cancer cell lines using the NCI (USA) procedure, and the % growth was minimally impacted, indicating telomerase enzyme selectivity. To investigate the interaction of compound 36b with the telomerase allosteric binding site, molecular docking and molecular dynamics simulations were used.

Mechanisms of the Antiproliferative and Antitumor Activity of Novel Telomerase-Carbonic Anhydrase Dual-Hybrid Inhibitors.

Human (h) telomerase (TL; EC 2.7.7.49) plays a key role in sustaining cancer cells by means of elongating telomeric repeats at the 3' ends of chromosomes. Since TL-inhibitor (TI) stand-alone cancer therapy has been proven to be remarkably challenging, a polypharmacological approach represents a valid alternative. Here we consider a series of compounds able to inhibit both hTL and the tumor-associated carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII. Compounds 7 and 9 suppressed hTL activity in both cell lysates and human colon cancer cell lines, and prolonged incubation with either 7 or 9 resulted in telomere shortening, cell cycle arrest, replicative senescence, and apoptosis. Enzyme kinetics showed that 7 and 9 are mixed-type inhibitors of the binding of DNA primers and deoxynucleoside triphosphate (dNTP) to the TL catalytic subunit hTERT, which is in agreement with docking experiments. Compound 9 showed antitumor activity in Colo-205 mouse xenografts and suppressed telomerase activity by telomere reduction.

The Introduction of Hydrazone, Hydrazide, or Azepane Moieties to the Triterpenoid Core Enhances an Activity Against M. tuberculosis.

BACKGROUND: Triterpenoids exhibit a wide spectrum of antimicrobial activity. OBJECTIVE: The objective of this study was to synthesize a series of nitrogen derivatives based on lupane, oleanane, and ursane triterpenoids with high antitubercular activity. METHODS: Isonicotinoylhydrazones were prepared via the reaction of 3-oxotriterpenic acids or betulonic aldehyde with isoniazid (INH) in yields of 54-72%. N-Acylation of betulonic or azepanobetulinic acids led to lupane C28 hydrazides and dihydrazides. The derivatives were evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H37RV and single-drug resistance (SDR)-TB in the National Institute of Allergy and Infectious Diseases, USA. Molecular docking was performed to evaluate the possible binding modes of investigated compounds in the active site of Diterpene synthase (Rv3378c). RESULTS: The obtained compounds are represented by C3 or C28 conjugates with hydrazine hydrate or INH. Some compounds demonstrated from high minimum inhibitory concentration (MIC ≤ 10 µg/mL) to excellent (MICs from 0.19 to 1.25 µg/mL) activity against MTB H37RV. Two lupane conjugates with INH were the leading compounds against MTB H37RV and some SDR-strains with MICs ranged from 0.19 to 1.70 µg/mL. Molecular docking of active compounds to diterpene synthase showed that these moieties accommodate the active site of the enzyme. CONCLUSION: It was revealed that the conjugation of lupanes with INH at C3 is more effective than at C28 and the lupane skeleton is preferable among oleanane and ursane types. The replacement of native hexacarbocyclic A ring to seven-member azepane ring is favorably for inhibition of both MTB H37RV and SDR-strains. These data could possibly mean that the antitubercular activity against INH-resistant strains (INH-R) came from both triterpenoid and isoniazid parts of the hybrid molecules. Azepanobetulin showed the highest activity against both INH-R strains in comparison with other triterpenoids and INH. Thus, the introduction of hydrazone, hydrazide (dihydrazide), or azepane moieties into the triterpenoid core is a promising way for the development of new anti-tubercular agents.

Interactions of galeterone and its 3-keto-Δ4 metabolite (D4G) with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2).

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation.

Isatin (indole-2, 3-dione) is a non-peptide endogenous bioregulator exhibiting a wide spectrum of biological activity, realized in the cell via interactions with numerous isatin-binding proteins, their complexes, and (sub) interactomes. There is increasing evidence that isatin may be involved in the regulation of complex formations by modulating the affinity of the interacting protein partners. Recently, using Surface Plasmon Resonance (SPR) analysis, we have found that isatin in a concentration dependent manner increased interaction between two human mitochondrial proteins, ferrochelatase (FECH), and adrenodoxine reductase (ADR). In this study, we have investigated the affinity-enhancing effect of isatin on the FECH/ADR interaction. The SPR analysis has shown that FECH forms not only homodimers, but also FECH/ADR heterodimers. The affinity-enhancing effect of isatin on the FECH/ADR interaction was highly specific and was not reproduced by structural analogues of isatin. Bioinformatic analysis performed using three dimensional (3D) models of the interacting proteins and in silico molecular docking revealed the most probable mechanism involving FECH/isatin/ADR ternary complex formation. In this complex, isatin is targeted to the interface of interacting FECH and ADR monomers, forming hydrogen bonds with both FECH and ADR. This is a new regulatory mechanism by which isatin can modulate protein-protein interactions (PPI).

The interactions of a number of steroid-metabolizing cytochromes P450 with abiraterone D4A metabolite: spectral analysis and molecular docking.

The interactions of pharmacologically active 3-keto-Δ4-metabolite of anticancer drug abiraterone (D4A) with steroid-metabolizing cytochromes P450 (CYP51A1, CYP11A1, CYP19A1) was studied by absorption spectroscopy and molecular docking. Both abiraterone and D4A induce type I spectral changes of CYP51A1, one of the enzymes of cholesterol biosynthesis. We have revealed that D4A did not induce spectral changes of CYP11A1, the key enzyme of pregnenolone biosynthesis, unlike abiraterone (type II ligand of CYP11A1). On the contrary, D4A interacts with the active site of CYP19A1, the key enzyme of estrogen biosynthesis, inducing type II spectral changes, while abiraterone does not. Spectral analysis allowed us to calculate spectral dissociation constant (KS) for each complex of cytochrome P450 with respective ligands. The data were supported by molecular docking. The obtained results broaden understanding of interactions of D4A with some of the key steroid-metabolizing cytochromes P450 and allow one to predict possible disproportions of steroid metabolism.

Functional and structural features of proteins associated with alternative splicing.

The alternative splicing is a mechanism increasing the number of expressed proteins and a variety of these functions. We uncovered the protein domains most frequently lacked or occurred in the splice variants. Proteins presented by several isoforms participate in such processes as transcription regulation, immune response, etc. Our results displayed the association of alternative splicing with branched regulatory pathways. By considering the published data on the protein proteins encoded by the 18th human chromosome, we noted that alternative products display the differences in several functional features, such as phosphorylation, subcellular location, ligand specificity, protein-protein interactions, etc. The investigation of alternative variants referred to the protein kinase domain was performed by comparing the alternative sequences with 3D structures. It was shown that large enough insertions/deletions could be compatible with the kinase fold if they match between the conserved secondary structures. Using the 3D data on human proteins, we showed that conformational flexibility could accommodate fold alterations in splice variants. The investigations of structural and functional differences in splice isoforms are required to understand how to distinguish the isoforms expressed as functioning proteins from the non-realized transcripts. These studies allow filling the gap between genomic and proteomic data.

New steroidal oxazolines, benzoxazoles and benzimidazoles related to abiraterone and galeterone.

Seven new oxazoline, benzoxazole and benzimidazole derivatives were synthesized from 3ß-acetoxyandrosta-5,16-dien-17-carboxylic, 3ß-acetoxyandrost-5-en-17ß-carboxylic and 3ß-acetoxypregn-5-en-21-oic acids. Docking to active site of human 17α-hydroxylase/17,20-lyase revealed that all oxazolines, as well as benzoxazoles and benzimidazoles comprising Δ16 could form stable complexes with enzyme, in which steroid moiety is positioned similarly to that of abiraterone and galeterone, and nitrogen atom coordinates heme iron, while 16,17-saturated benzoxazoles and benzimidazoles could only bind in a position where heterocycle is located nearly parallel to heme plane. Modeling of the interaction of new benzoxazole and benzimidazole derivatives with androgen receptor revealed the destabilization of helix 12, constituting activation function 2 (AF2) site, by mentioned compounds, similar to one induced by known antagonist galeterone. The synthesized compounds inhibited growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole and 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-benzimidazole was superior and could inspire further investigations of these compounds as potential anti-cancer agents.

Estimation of the inhibiting impact of abiraterone D4A metabolite on human steroid 21-monooxygenase (CYP21A2).

Abiraterone D4A metabolite, the product of 3ß-hydroxysteroid dehydrogenase activity toward abiraterone, may serve as a potential antitumor agent for the treatment of prostate cancer. The main adverse effect of abiraterone is the disruption of corticosteroid biosynthesis, and the more pharmacologically active abiraterone D4A metabolite may have the same issues. We therefore estimated the inhibiting impact of the abiraterone D4A metabolite on one of the key corticosteroidogenic enzymes - human steroid 21-monooxygenase (CYP21A2). Molecular docking of D4A into the active site of CYP21A2 has been predicted to be similar to abiraterone binding with the enzyme. Abiraterone D4A metabolite, similar to abiraterone, induces type II spectral changes of CYP21A2. The spectral dissociation constant for the abiraterone D4A metabolite-CYP21A2 complex was calculated as 3.4 ±â€¯0.5 µM. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8 ±â€¯0.8 µM, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.

In vitro interactions of abiraterone, erythromycin, and CYP3A4: implications for drug-drug interactions.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 µM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki  = 8.1 ± 1.2 µM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.

The impact of long-distance mutations on the Ω-loop conformation in TEM type ß-lactamases.

ß-lactamases are hydrolytic enzymes primarily responsible for occurrence and abundance of bacteria resistant to ß-lactam antibiotics. TEM type ß-lactamases are formed by the parent enzyme TEM-1 and more than two hundred of its mutants. Positions for the known amino acid substitutions cover ∼30% of TEM type enzyme's sequence. These substitutions are divided into the key mutations that lead to changes in catalytic properties of ß-lactamases, and the secondary ones, which role is poorly understood. In this study, Residue Interaction Networks were constructed from molecular dynamic trajectories of ß-lactamase TEM-1 and its variants with two key substitutions, G238S and E240K, and their combinations with secondary ones (M182T and Q39K). Particular attention was paid to a detailed analysis of the interactions that affect conformation and mobility of the Ω-loop, representing a part of the ß-lactamase active site. It was shown that key mutations weakened the stability of contact inside the Ω-loop thus increasing its mobility. Combination of three amino acid substitutions, including the 182 residue, leads to the release of R65 promoting its new contacts with N175 and D176. As a result, Ω-loop is fixed on the protein globule. The second distal mutation Q39K prevents changes in spatial position of R65, which lead to the weakening of the effect of M182T substitution and the recovery of the Ω-loop mobility. Thus, the distal secondary mutations are directed for recovering the mobility of enzyme disturbed by the key mutations responsible for expansion of substrate specificity. AbbreviationsESBLextended spectrum beta-lactamasesIRinhibitor resistant beta-lactamasesMDmolecular dynamicsRINresidue interaction networksRMSDroot mean square deviationRMSFroot mean square fluctuations.Communicated by Ramaswamy H. Sarma.

The Role of the Ω-Loop in Regulation of the Catalytic Activity of TEM-Type ß-Lactamases.

Bacterial resistance to ß-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed ß-lactamases (ßLs). The evolution of serine-class A ß-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme's active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM-type ß-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl-enzyme complex and the hydrolysis of the ß-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type ß-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of ß-lactamases.