Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

Supramolecular Hydrogels and Water Channels of Differing Diameters from Dipeptide Isomers.

Dipeptides stereoisomers and regioisomers composed of norleucine (Nle) and phenylalanine (Phe) self-assemble into hydrogels under physiological conditions that are suitable for cell culture. The supramolecular behavior, however, differs as the packing modes comprise amphipathic layers or water channels, whose diameter is defined by either four or six dipeptide molecules. A variety of spectroscopy, microscopy, and synchrotron-radiation-based techniques unveil fine details of intermolecular interactions that pinpoint the relationship between the chemical structure and ability to form supramolecular architectures that define soft biomaterials.

Unlocking Amides: A General Method for the Self-Immolative Release of Amide-Containing Molecules.

The controlled liberation of molecules from a constructed framework is a subject of profound interest across various chemical fields. It allows for the masking of a molecule's properties and precise deployment upon a single controllable release event. While numerous methodologies have been developed for amines, alcohols, and thiols, approaches for utilising amides as payload-release handles are still in their early stages of development, despite the prevalence of amides in therapeutic compounds and materials. Herein, is presented a comprehensive strategy for the controlled and selective release of a diverse range of amides with stable linkers. The versatility of this approach is demonstrated by its successful application in the targeted release of various amide-containing drugs in their natural form via the use of commonly used trigger motifs, such as dipeptides or glycosides. As a proof of concept, the FDA-approved antibiotic linezolid has been successfully converted into a prodrug form and released selectively only in the presence of the trigger event. Significantly, in its prodrug state, no activity against Mycobacterium tuberculosis was exhibited. Linezolid's full potential was achieved only upon controlled release, where an equipotent efficacy to the free linezolid control was demonstrated, thus emphasising the immense potential of this method.

New origin of life theory may explain biomolecular handedness.

Experiments suggest chemical reaction rates explain how proteins came to be built from left-handed building blocks.

Cucurbit[8]uril Binds Nonterminal Dipeptide Sites with High Affinity and Induces a Type II ß-Turn.

In an effort to target polypeptides at nonterminal sites, we screened the binding of the synthetic receptor cucurbit[8]uril (Q8) to a small library of tetrapeptides, each containing a nonterminal dipeptide binding site. The resulting leads were characterized in detail using a combination of isothermal titration calorimetry, 1H NMR spectroscopy, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and X-ray crystallography. The equilibrium dissociation constant values determined for the binding of Q8 to nonterminal dipeptide sites Lys-Phe (KF) and Phe-Lys (FK) were 60 and 86 nm, respectively. These are to the best of our knowledge the highest affinities reported to date for any synthetic receptor targeting a nonterminal site on an unmodified peptide. A 0.79 Å resolution crystal structure was obtained for the complex of Q8 with the peptide Gly-Gly-Leu-Tyr-Gly-Gly-Gly (GGLYGGG) and reveals structural details of the pair-inclusion motif. The molecular basis for recognition is established to be the inclusion of the side chains of Leu and Tyr residues, as well as an extensive network of hydrogen bonds between the peptide backbone, the carbonyl oxygens of Q8, and proximal water molecules. In addition, the crystal structure reveals that Q8 induces a type II ß-turn. The sequence-selectivity, high affinity, reversibility, and detailed structural characterization of this system should facilitate the development of applications involving ligand-induced polypeptide folding.

Spatiotemporal modeling of radiopharmaceutical transport in solid tumors: Application to 177Lu-PSMA therapy of prostate cancer.

BACKGROUND AND OBJECTIVE: 177Lu-labeled prostate-specific membrane antigen (PSMA) radiopharmaceutical therapy (RPT) represents a pivotal advancement in addressing prostate cancer. However, existing therapies, while promising, remain incompletely understood and optimized. Computational models offer potential insights into RPTs, aiding in clinical drug delivery enhancement. In this study, we investigate the impact of various physiological parameters on the delivery of 177Lu-PSMA-617 RPT using the convection-diffusion-reaction (CDR) model. METHODS: Our investigation encompasses tumor geometry and surrounding tissue, characterized by well-defined boundaries and initial conditions. Utilizing the finite element method, we solve governing equations across a range of parameters: dissociation constant KD (1, 0.1, 0.01 [nM]), internalization rate (0.01-0.0001 [min-1]), diverse tumor shapes, and variable necrotic zone sizes. This model can provide an accurate analysis of radiopharmaceutical delivery from the injection site to the tumor cell, including drug transport in the vascular, interstitial, and intracellular spaces, and considering important parameters (e.g., drug extravasation from microvessels or to lymphatic vessels, the extracellular matrix, receptors, and intracellular space). RESULTS: Our findings reveal significant enhancements in tumor-absorbed doses as KD decreases. This outcome can be attributed to the higher affinity of radiopharmaceuticals for PSMA receptors as KD diminishes, facilitating a more efficient binding and retention of the therapeutic agent within the tumor microenvironment. Additionally, tumor-absorbed doses for KD ∼ 1 [nM] show an upward trend with higher internalization rates. This observation can be rationalized by considering that a greater internalization rate would result in a higher proportion of radiopharmaceuticals being taken up by tumor cells after binding to receptors on the cell surface. Notably, tumor shape and necrotic zone size exhibit limited influence on tumor absorbed dose. CONCLUSIONS: The present study employs the CDR model to explore the role of physiological parameters in shaping 177Lu-PSMA-617 RPT delivery. These findings provide insights for improving prostate cancer therapy by understanding radiopharmaceutical transport dynamics. This computational approach contributes to advancing our understanding of radiopharmaceutical delivery mechanisms and has implications for enhancing treatment efficacy.

Three Stages of Dynamic Assembly Process of Dipeptide-Based Supramolecular Gel Revealed by In Situ Infrared Spectroscopy.

The exploration of short peptide-based assembly is vital for understanding protein-misfolding-associated diseases and seeking strategies to attenuate aggregate formation. While, the molecular mechanism of their structural evolution remains poorly studied in view of the dynamic and unpredictable assembly process. Herein, infrared (IR) spectroscopy, which serves as an in situ and real-time analytical technique, was intelligently employed to investigate the mechanism of phase transition and aggregate formation during the dynamic assembly process of diphenylalanine. Combined with other spectroscopy and electron microscopy technologies, three stages of gel formation and the main driving forces in different stages were revealed. A variety of stoichiometric methods such as continuous wavelet transform, principal component analysis, and two-dimensional correlation spectroscopy techniques were conducted to analyze the original time-dependent IR spectra to obtain detailed information on the changes in the amide bands and hydration layer. The microenvironment of hydrogen bonding among amide bands was significantly changed with the addition of pyridine derivatives, resulting in great differences in the properties of co-assembled gels. This work not only provides a universal analytical way to reveal the dynamic assembly process of dipeptide-based supramolecular gel but also expands their applications in supramolecular regulation and high-throughput screens in situ.

C9orf72-associated dipeptide protein repeats form A11-positive oligomers in amyotrophic lateral sclerosis and frontotemporal dementia.

Hexanucleotide repeat expansion in C9orf72 is one of the most common causes of amyotrophic lateral sclerosis and frontotemporal dementia. The hexanucleotide expansion, formed by GGGGCC (G4C2) repeats, leads to the production of five dipeptide protein repeats (DPRs) via repeat-associated non-AUG translation. Among the five dipeptide repeats, Gly-Arg, Pro-Arg, and Gly-Ala form neuronal inclusions that contain aggregates of the peptides. Several studies have attempted to model DPR-associated toxicity using various repeat lengths, which suggests a unique conformation that is cytotoxic and is independent of the repeat length. However, the structural characteristics of DPR aggregates have yet to be determined. Increasing evidence suggests that soluble species, such as oligomers, are the main cause of toxicity in proteinopathies, such as Alzheimer's and Parkinson's disease. To investigate the ability of DPRs to aggregate and form toxic oligomers, we adopted a reductionist approach using small dipeptide repeats of 3, 6, and 12. This study shows that DPRs, particularly glycine-arginine and proline-arginine, form oligomers that exhibit distinct dye-binding properties and morphologies. Importantly, we also identified toxic DPR oligomers in amyotrophic lateral sclerosis and frontotemporal dementia postmortem brains that are morphologically similar to those generated recombinantly. This study demonstrates that, similar to soluble oligomers formed by various amyloid proteins, DPR oligomers are toxic, independent of their repeat length.

Interrupted Polonovski Strategy for the Synthesis of Functionalized Amino Acids and Peptides.

The α-functionalization of carbamate-protected hydroxylamine glycine derivatives, acting as imine surrogates via an interrupted Polonovski reaction, is described to access functionalized amino acid derivatives. The addition of C, N, O, and S nucleophiles was achieved in a one-pot procedure in 37% to 92% yield. This method could be extended to dipeptide derivatives for the functionalization of both the C-terminus and N-terminus.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

Self-assembly of heterochiral, aliphatic dipeptides with Leu.

This work describes the self-assembly behavior of heterochiral, aliphatic dipeptides, l-Leu-d-Xaa (Xaa = Ala, Val, Ile, Leu), in green solvents such as acetonitrile (MeCN) and buffered water at neutral pH. Interestingly, water plays a structuring role because at 1% v/v, it enables dipeptide self-assembly in MeCN to yield organogels, which then undergo transition towards crystals. Other organic solvents and oils were tested for gelation, and metastable gels were formed in tetrahydrofuran, although at high peptide concentration (80 mM). Single-crystal X-ray diffraction revealed the dipeptides' supramolecular packing modes in amphipathic layers, as opposed to water channels reported for the homochiral Leu-Leu, or hydrophobic columns reported for homochiral Leu-Val and Leu-Ile.

Representation of Protein Dynamics Disentangled by Time-Structure-Based Prior.

Representation learning (RL) is a universal technique for deriving low-dimensional disentangled representations from high-dimensional observations, aiding in a multitude of downstream tasks. RL has been extensively applied to various data types, including images and natural language. Here, we analyze molecular dynamics (MD) simulation data of biomolecules in terms of RL. Currently, state-of-the-art RL techniques, mainly motivated by the variational principle, try to capture slow motions in the representation (latent) space. Here, we propose two methods based on an alternative perspective on the disentanglement in the latent space. By disentanglement, we here mean the separation of underlying factors in the simulation data, aiding in detecting physically important coordinates for conformational transitions. The proposed methods introduce a simple prior that imposes temporal constraints in the latent space, serving as a regularization term to facilitate the capture of disentangled representations of dynamics. Comparison with other methods via the analysis of MD simulation trajectories for alanine dipeptide and chignolin validates that the proposed methods construct Markov state models (MSMs) whose implied time scales are comparable to those of the state-of-the-art methods. Using a measure based on total variation, we quantitatively evaluated that the proposed methods successfully disentangle physically important coordinates, aiding the interpretation of folding/unfolding transitions of chignolin. Overall, our methods provide good representations of complex biomolecular dynamics for downstream tasks, allowing for better interpretations of the conformational transitions.

LPATH: A Semiautomated Python Tool for Clustering Molecular Pathways.

The pathways by which a molecular process transitions to a target state are highly sought-after as direct views of a transition mechanism. While great strides have been made in the physics-based simulation of such pathways, the analysis of these pathways can be a major challenge due to their diversity and variable lengths. Here, we present the LPATH Python tool, which implements a semiautomated method for linguistics-assisted clustering of pathways into distinct classes (or routes). This method involves three steps: 1) discretizing the configurational space into key states, 2) extracting a text-string sequence of key visited states for each pathway, and 3) pairwise matching of pathways based on a text-string similarity score. To circumvent the prohibitive memory requirements of the first step, we have implemented a general two-stage method for clustering conformational states that exploits machine learning. LPATH is primarily designed for use with the WESTPA software for weighted ensemble simulations; however, the tool can also be applied to conventional simulations. As demonstrated for the C7eq to C7ax conformational transition of the alanine dipeptide, LPATH provides physically reasonable classes of pathways and corresponding probabilities.

Piezoactive Bioorganic Diphenylalanine Films: Mechanism of Phase Formation.

Self-organized peptides are unique materials with various applications in biology, medicine, and nanotechnology. Many of these applications require fabrication of homogeneous thin films having high piezoelectric effect and sufficiently low roughness. Recently, a facile method for the controlled deposition of flat solid films of the most studied peptide, diphenylalanine (FF), has been proposed, which is based on the crystallization of FF in the amorphous phase under the action of water vapor. This method is very advantageous compared with crystallization from a liquid phase reported previously. Here, we thoroughly investigate the mechanism of solid-state transformation from the amorphous to crystalline phase. The study revealed that the process proceeds in two distinct stages, maintaining clamped condition of self-assembling building blocks that preserve the films' morphology and high piezoelectric activity. We emphasize the critical role of water diffusion that governs two-dimensional growth of crystalline domains in FF films, merging in very dense, flat, and homogeneous films. These findings open a wide perspective for using this methodology for the direct fabrication of biofilms from the amorphous phase. We thus expect the application of these films to various nanotechnological applications of self-assembled structures.

Tunable Chirality of Self-Assembled Dipeptides Mediated by Bipyridine Derivative.

Supramolecular peptide assemblies have been widely used for the development of biomedical, catalytical, and optical materials with chiral nanostructures in view of the intrinsic chirality of peptides. However, the assembly pathway and chiral transformation behavior of various peptides remain largely elusive especially for the transient assemblies under out-of-equilibrium conditions. Herein, the N-fluorenylmethoxycarbonyl-protected phenylalanine-tyrosine dipeptide (Fmoc-FY) was used as a peptide assembly platform, which showed that the assembly proceeds multistep evolution. The original spheres caused by liquid-liquid phase separation (LLPS) can nucleate and elongate into the formation of right-handed helices which were metastable and easily converted into microribbons. Interestingly, a bipyridine derivative can be introduced to effectively control the assembly pathway and induce the formation of thermodynamically stable right-handed or left-handed helices at different stoichiometric ratios. In addition, the chiral assembly can also be regulated by ultrasound or enzyme catalysis. This minimalistic system not only broadens the nucleation-elongation mechanisms of protein aggregates but also promotes the controllable design and development of chiral biomaterials.

A Desmethylphosphinothricin Dipeptide Derivative Effectively Inhibits Escherichia coli and Bacillus subtilis Growth.

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-PH), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-PH (L-Leu-D,L-Glu-γ-PH). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-PH, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-PH had a significantly improved antibacterial activity (2 µg/mL), at a concentration between the non-desmethytaled (0.1 µg/mL) and the phosphonic (80 µg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-PH displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.

Evaluation of glycyl-arginine and lysyl-aspartic acid dipeptides for their antimicrobial, antibiofilm, and anticancer potentials.

Antibacterial resistance and cancer are worldwide challenges and have been defined as major threats by international health organizations. Peptides are produced naturally by all organisms and have a variety of immunomodulatory, physiological, and wound-healing properties. They can also provide protection against microorganisms and tumor cells. Therefore, we aimed to determine the antimicrobial, antibiofilm, and anticancer potentials of Glycyl-Arginine and Lysyl-Aspartic acid dipeptides. The Broth Dilution and Crystal Violet Binding assays assessed the antimicrobial tests and biofilm inhibitory effects. The MTT assay was used to measure the cytotoxic effects of dipeptides on HeLa cell viability. According to our results, Candida tropicalis T26 and Proteus mirabilis U15 strains were determined as more resistant to Staphylococcus epidermidis W17 against Glycyl-Arginine and Lysyl-Aspartic acid dipeptides with MICs higher than 2 mM (1 mg/mL). Sub-MICs of Glycyl-Arginine caused inhibitions against biofilm formation of all the tested clinical isolates, with the highest inhibition observed against S. epidermidisW17. Lysyl-Aspartic acid exhibited zero to no effect against biofilm formation of P. mirabilisU15, and S. epidermidisW17, whereas it exhibited 52% inhibition of biofilm formation of C. tropicalisT26. Cell viability results revealed that HeLa cell viability decreases with increasing concentration of both dipeptides. Also, parallel to antimicrobial tests, Glycyl-Arginine has a greater cytotoxic effect compared to Lysyl-Aspartic acid. The findings from this study will contribute to the advancement of novel strategies involving dipeptide-based synthesizable molecules and drug development studies. However, it is essential to note that there are still challenges, including the need for extensive experimental and clinical trials.

Investigating the complexation propensity of self-assembling dipeptides with the anticancer peptide-drug Bortezomib: a computational study.

The investigation of potential self-assembled peptides as carriers for the delivery of anticancer drug Bortezomib is the topic of the present study. The self-assembly of Bortezomib in water is examined using all-atom molecular dynamics simulations and corresponding experimental results from FESEM experiments. In addition, a series of dipeptides with a similar chemical formula to Bortezomib with hydrogel-forming ability are being investigated for their propensity to bind to the drug molecule. Dipeptides are divided into two classes, the protected FF (Fmoc-FF and Z-FF) and the LF-based (Cyclo-LF and LF) ones. The thermodynamic stability of the complexes formed in an aqueous environment, as well as key morphological features of the nanoassemblies are investigated at the molecular level. Binding enthalpy between Bortezomib and dipeptides follows the increasing order: LF < Cyclo-LF < Fmoc-FF < Z-FF under both van der Waals and electrostatic contributions. Protected FF dipeptides have a higher affinity for the drug molecule, which will favor its entrapment, giving them an edge over the LF based dipeptides. By evaluating the various measures, regarding both the binding between the two components and the eventual ability of controlled drug release, we conclude that the protected FF class is a more suitable candidate for drug release of Bortezomib, whereas among its two members, Fmoc-FF appears to be more promising. The selection of the optimal candidates based on the present computational study will be a stepping stone for future detailed experimental studies involving the encapsulation and controlled release of Bortezomib both in vitro and in vivo.

Integrative generalized master equation: A method to study long-timescale biomolecular dynamics via the integrals of memory kernels.

The generalized master equation (GME) provides a powerful approach to study biomolecular dynamics via non-Markovian dynamic models built from molecular dynamics (MD) simulations. Previously, we have implemented the GME, namely the quasi Markov State Model (qMSM), where we explicitly calculate the memory kernel and propagate dynamics using a discretized GME. qMSM can be constructed with much shorter MD trajectories than the MSM. However, since qMSM needs to explicitly compute the time-dependent memory kernels, it is heavily affected by the numerical fluctuations of simulation data when applied to study biomolecular conformational changes. This can lead to numerical instability of predicted long-time dynamics, greatly limiting the applicability of qMSM in complicated biomolecules. We present a new method, the Integrative GME (IGME), in which we analytically solve the GME under the condition when the memory kernels have decayed to zero. Our IGME overcomes the challenges of the qMSM by using the time integrations of memory kernels, thereby avoiding the numerical instability caused by explicit computation of time-dependent memory kernels. Using our solutions of the GME, we have developed a new approach to compute long-time dynamics based on MD simulations in a numerically stable, accurate and efficient way. To demonstrate its effectiveness, we have applied the IGME in three biomolecules: the alanine dipeptide, FIP35 WW-domain, and Taq RNA polymerase. In each system, the IGME achieves significantly smaller fluctuations for both memory kernels and long-time dynamics compared to the qMSM. We anticipate that the IGME can be widely applied to investigate biomolecular conformational changes.

N,O-Benzylidene Acetal Dipeptides (NBDs) Enable the Synthesis of Difficult Peptides via a Kinked Backbone Strategy.

Proteins with highly hydrophobic regions or aggregation-prone sequences are typically difficult targets for chemical synthesis at the current stage, as obtaining such type of peptides via solid-phase peptide synthesis requires sophisticated operations. Herein, we report N,O-benzylidene acetal dipeptides (NBDs) as robust and effective building blocks to allow the direct synthesis of difficult peptides and proteins via a kinked backbone strategy. The effectiveness and easy accessibility of NBDs have been well demonstrated in our chemical syntheses of various challenging peptides and proteins, including chemokine, therapeutic hormones, histone, and glycosylated erythropoietin.