Extensive targeting of chemical space at the prime side of ketoamide inhibitors of rhomboid proteases by branched substituents empowers their selectivity and potency.

Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipidic cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesized here containing a branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2.

A Direct Route to closo-SB n Cl n Thiaboranes from Simple Electron-Precise Synthons: The Different Role of Chalcogen Bonding in SB5Cl5 and SB11Cl11 Crystals.

Six-vertex closo-SB5Cl5 (1) and ten-vertex closo-1-SB9Cl9 (2) thiaboranes have been prepared, besides the already known 12-vertex closo-SB11Cl11 (3), from the co-pyrolysis reaction of B2Cl4 with S2Cl2 at 280 ºC in vacuo. The compounds are sublimable, off-white solids. Their elemental composition has been determined by high-resolution mass spectrometry. They were further characterized by one- and two-dimensional 11B-spectroscopy and X-ray structure determination for 1 and 3. Ab initio/GIAO/NMR computations support octahedral, bicapped square-antiprismatic, and icosahedral geometries for 1, 2 and 3, respectively, as expected based on their closo-electron counts. 1 is the first isolated example of a neutral polyhedral closo-thiaborane with a cluster size smaller than ten vertices. The solid-state structure of 3 is one of the rare examples of a single-crystal X-ray structure determination of an icosahedral heteroborane reported. The corresponding crystal-packing forces show the different role of chalcogen bonding in these octahedral and icosahedral crystals. In addition, there is a mass-spectroscopy evidence for the recurrent formation of further thiaborane homologs of closo-SBnCln with n = 4, 6, 10, and supra-icosahedral 12.

Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

SQM2.20: Semiempirical quantum-mechanical scoring function yields DFT-quality protein-ligand binding affinity predictions in minutes.

Accurate estimation of protein-ligand binding affinity is the cornerstone of computer-aided drug design. We present a universal physics-based scoring function, named SQM2.20, addressing key terms of binding free energy using semiempirical quantum-mechanical computational methods. SQM2.20 incorporates the latest methodological advances while remaining computationally efficient even for systems with thousands of atoms. To validate it rigorously, we have compiled and made available the PL-REX benchmark dataset consisting of high-resolution crystal structures and reliable experimental affinities for ten diverse protein targets. Comparative assessments demonstrate that SQM2.20 outperforms other scoring methods and reaches a level of accuracy similar to much more expensive DFT calculations. In the PL-REX dataset, it achieves excellent correlation with experimental data (average R2 = 0.69) and exhibits consistent performance across all targets. In contrast to DFT, SQM2.20 provides affinity predictions in minutes, making it suitable for practical applications in hit identification or lead optimization.

Chlorinated polyhedral selenaboranes revisited by joint experimental/computational efforts: the formation of closo-1-SeB9Cl9 and the crystal structure of closo-SeB11Cl11.

The recent success in the formation of chlorinated telluraboranes and the reactivities of pnictogenaboranes prompted us to re-examine the vacuum co-pyrolysis of B2Cl4 with Se2Cl2 at various molar ratios and temperatures in order to search for the generation of other polyhedral selenaboranes than closo-SeB5Cl5 (1a) and closo-SeB11Cl11 (1b), the latter being observed earlier. Interestingly, a new compound with the elemental composition SeB9Cl9 (2) was detected, this time by high- and low-resolution mass spectrometry. Further characterization by 1- and 2-D 11B-NMR spectroscopy suggests that 2 should adopt a closed bicapped square-antiprismatic geometry with selenium at the apical position. Moreover, vacuum sublimation gave suitable crystals of 1b, which were subjected to single-crystal X-ray structure determination. Crystallographic data analysis confirmed that 1b, consistent with its 26 skeletal electron count, adopts a distorted icosahedral structure close to the symmetry of C5v. Computations at the DFT-D3 level have revealed that 33% of the total computed binding motifs in the grown 1b crystals are due to the very strong chalcogen bonding. Moreover, SAPT decomposition has shown that the bonding motifs in the crystals are stabilized mainly by dispersion and electrostatic terms. Homodecoupling and high resolution 11B NMR and 77Se NMR experiments have resolved both coupling constants 1J(11B11B) and 1J(77Se11B) as well as the 77Se chemical shift of 1a and 1b, which are in reasonable agreement with the corresponding computed values. The computed 11B chemical shifts of 2 were determined by the well-established DFT/GIAO/NMR structural tool based on its B3LYP/6-311+G** internal coordinates. They agree well with the experimental values and provide a good representation of the molecular structure of 2 in solution. The extraordinary downfield 11B NMR chemical shift of B(10) in 2 has been ascribed to the intensive paramagnetic contribution to the shielding tensor in this bicapped square-antiprismatic motif. Calculations of the synproportionation free energies of smaller (n - 1) closo-selenaboranes with larger-sized (n + 1) ones support the extraordinary stability of octahedral, bicapped square-antiprismatic and icosahedral closo motifs in the SeBnCln family (n = 4-12).

Boron-based octahedral dication experimentally detected: DFT surface confirms its availability.

Borane and heteroborane clusters have been known as neutral or anionic species. In contrast to them, several ten-vertex monocationic nido and closo dicarbaborane-based systems have recently emerged from the reaction of the parent bicapped-square antiprismatic dicarbaboranes with N-heterocyclic carbenes followed by the protonization of the corresponding nido intermediates. The expansion of these efforts has afforded the very first closo-dicationic octahedral phosphahexaborane along with new closo-monocationic pnictogenahexaboranes of the same shapes. All are the products of the one-pot procedure that consists in the reaction of the same carbenes with the parent closo-1,2-Pn2B4Br4 (Pn = As, P). Whereas in the case of phosphorus such a monocation appears to be a mixture of stable intermediates, and arsenahexaboranyl monocation has occurred as the final product, all of them without using any subsequent reaction. The well-established DFT/ZORA/NMR approach has unambiguously confirmed the existence of these species in solution, and computed electrostatic potentials have revealed the delocalization of the positive charge in these monocations and in the very first dication, namely within the octahedral shapes in both cases.

DFT Surface Infers Ten-Vertex Cationic Carboranes from the Corresponding Neutral closo Ten-Vertex Family: The Computed Background Confirming Their Experimental Availability.

Modern computational protocols based on the density functional theory (DFT) infer that polyhedral closo ten-vertex carboranes are key starting stationary states in obtaining ten-vertex cationic carboranes. The rearrangement of the bicapped square polyhedra into decaborane-like shapes with open hexagons in boat conformations is caused by attacks of N-heterocyclic carbenes (NHCs) on the closo motifs. Single-point computations on the stationary points found during computational examinations of the reaction pathways have clearly shown that taking the "experimental" NHCs into account requires the use of dispersion correction. Further examination has revealed that for the purposes of the description of reaction pathways in their entirety, i.e., together with all transition states and intermediates, a simplified model of NHCs is sufficient. Many of such transition states resemble in their shapes those that dictate Z-rearrangement among various isomers of closo ten-vertex carboranes. Computational results are in very good agreement with the experimental findings obtained earlier.

Strategies for the Design of PEDOT Analogues Unraveled: the Use of Chalcogen Bonds and σ-Holes.

In this theoretical study, we set out to demonstrate the substitution effect of PEDOT analogues on planarity as an intrinsic indicator for electronic performance. We perform a quantum mechanical (DFT) study of PEDOT and analogous model systems and demonstrate the usefulness of the ωB97X-V functional to simulate chalcogen bonds and other noncovalent interactions. We confirm that the chalcogen bond stabilizes the planar conformation and further visualize its presence via the electrostatic potential surface. In comparison to the prevalent B3LYP, we gain 4-fold savings in computational time and simulate model systems of up to a dodecamer. Implications for design of conductive polymers can be drawn from the results, and an example for self-doped polymers is presented where modulation of the strength of the chalcogen bond plays a significant role.

The Telluraboranes closo-TeB5 Cl5 and closo-TeB11 Cl11 with Exceptionally Long Body Diagonals: Synthetic and Bonding Motifs for Innovative Octahedral and Icosahedral Geometries.

Six-vertex closo-TeB5 Cl5 (1) and twelve-vertex closo-TeB11 Cl11 (2) telluraboranes have been prepared via co-pyrolysis of B2 Cl4 with TeCl4 in vacuo at temperatures between 360 °C and 400 °C. Both compounds are sublimable, off-white solids, and they have been characterized by one- and two-dimensional 11 B NMR and high-resolution mass spectroscopy. Both ab initio/GIAO/NMR and DFT/ZORA/NMR computations support octahedral and icosahedral geometries for 1 and 2, respectively, as expected due to their closo-electron counts. The octahedral structure of 1 has been confirmed by single-crystal X-ray diffraction on an incommensurately modulated crystal. The corresponding bonding properties have been analyzed in terms of the intrinsic bond orbital (IBO) approach. 1 is the first example of a polyhedral telluraborane with a cluster size smaller than 10 vertices.

B-H⋯π and C-H⋯π interactions in protein-ligand complexes: carbonic anhydrase II inhibition by carborane sulfonamides.

Among non-covalent interactions, B-H⋯π and C-H⋯π hydrogen bonding is rather weak and less studied. Nevertheless, since both can affect the energetics of protein-ligand binding, their understanding is an important prerequisite for reliable predictions of affinities. Through a combination of high-resolution X-ray crystallography and quantum-chemical calculations on carbonic anhydrase II/carborane-based inhibitor systems, this paper provides the first example of B-H⋯π hydrogen bonding in a protein-ligand complex. It shows that the B-H⋯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-H⋯π interaction is twice as strong, with a slightly smaller contribution of dispersion and a slightly higher contribution of electrostatics. Such a detailed insight will facilitate the rational design of future protein ligands, controlling these types of non-covalent interactions.

Reactivity of Perhalogenated Octahedral Phospha- and Arsaboranes toward THF: A Joint Experimental/Computational Study.

Reactions of the perhalogenated polyhedral pnictogenaboranes closo-1,2-Pn2B4Hal4 (Pn = P, As; Hal = Cl, Br) with Lewis bases are presently being studied with a focus on rationalizing the sites of nucleophilic attacks on clusters bearing σ-holes. These σ-holes are localized both on pnictogens and, for Hal = Br, on bromine atoms, as revealed by electrostatic potential (ESP) and intrinsic bond orbital (IBO) analyses. Surprisingly, the attack of the cyclic ether THF on closo-1,2-Pn2B4Br4 does not occur on the site with the largest positive partial charge, centered in the middle of the pnictogen-pnictogen vector. Instead, presumably promoted by the positivated bromine substituents, THF inserts into the boron-bromine bonds of the negatively charged boron atoms opposite to the pnictogen atoms to form 4-(4-bromobut-1-oxy)-closo-1,2-Pn2B4Br3 (1-PB and 1-AsB) and 4,6-(4-bromobut-1-oxy)2-closo-1,2-Pn2B4Br2 (2-PB and 2-AsB). 11B and 31P chemical shift computations at various levels support the assignments of the signals, which reflect the correctness of the molecular geometries in solutions. The Lewis-acidic perchlorinated analogues closo-1,2-P2B4Cl4, closo-1,2-As2B4Cl4, and the mixed closo-1,2-AsPB4Cl4 bear negative charges. These negative charges are revealed by the Vs,max values when computing the electrostatic potentials both on the boron and the chlorine atoms. Due to this negative charge, the analogues do not react with THF unless they are heated above 66 °C, where they slowly decompose to borate esters B(OR)3 without the formation of concrete intermediates. The evaluation of 31P NMR data of 1-PB has allowed the experimental determination of the coupling constant 1J(31P(1), 31P(2)) = |143| Hz in a closo-diphosphaborane for the first time, which agrees well with the computed value of -178 Hz. The pioneering joint experimental vs computational interpretation of 31P NMR spectra in the area of boron cluster chemistry was decisive for the structural characterization of 1-PB and 2-PB.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-ß-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Access to cationic polyhedral carboranes via dynamic cage surgery with N-heterocyclic carbenes.

Polyhedral boranes and heteroboranes appear almost exclusively as neutral or anionic species, while the cationic ones are protonated at exoskeletal heteroatoms or they are instable. Here we report the reactivity of 10-vertex closo-dicarbadecaboranes with one or two equivalents of N-heterocyclic carbene to 10-vertex nido mono- and/or bis-carbene adducts, respectively. These complexes easily undergo a reaction with HCl to give cages of stable and water soluble 10-vertex nido-type cations with protonation in the form of a BHB bridge or 10-vertex closo-type cations containing one carbene ligand when originating from closo-1,10-dicarbadecaborane. The reaction of a 10-vertex nido mono-carbene adduct with phosphorus trichloride gives nido-11-vertex 2-phospha-7,8-dicarbaundecaborane, which undergoes an oxidation of the phosphorus atom to P = O, while the product of a bis-carbene adduct reaction is best described as a distorted C2B6H8 fragment bridged by the (BH)2PCl2+ moiety.

Thiaborane Icosahedral Barrier Increased by the Functionalization of all Terminal Hydrogens in closo-1-SB11H11.

The electrophilic substitution of icosahedral closo-1-SB11H11 with methyl iodide has resulted in two B-functionalized thiaboranes, 7,12-I2-2,3,4,5,6,8,9,10,11-(CH3)9-1-closo-SB11 and 7,8,12-I3-2,3,4,5,6,9,10,11-(CH3)8-closo-1-SB11, with the former being significantly predominant. These two icosahedral thiaboranes are the first cases of polysubstituted polyhedral boron clusters with another vertex that differs from B and C. Such polyfunctionalizations have increased the earlier observed thiaborane icosahedral barrier, not exhibiting any reactivity toward bases, unlike the parent thiaborane. The search for methylation pathways has revealed that the complete B11-methylation is impossible, like in the case of decaborane(14), where this seems to be a result of the positively charged upper parts of these two molecules.

The Structure-Based Design of SARS-CoV-2 nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors.

In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases (MTases), nsp14. This MTase catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to cap the guanosine triphosphate moiety of the newly synthesized viral RNA, yielding the methylated capped RNA and S-adenosyl-l-homocysteine (SAH). As the crystal structure of SARS-CoV-2 nsp14 is unknown, we have taken advantage of its high homology to SARS-CoV nsp14 and prepared its homology model, which has allowed us to identify novel SAH derivatives modified at the adenine nucleobase as inhibitors of this important viral target. We have synthesized and tested the designed compounds in vitro and shown that these derivatives exert unprecedented inhibitory activity against this crucial enzyme. The docking studies nicely explain the contribution of an aromatic part attached by a linker to the position 7 of the 7-deaza analogues of SAH.

Transformation of various multicenter bondings within bicapped-square antiprismatic motifs: Z-rearrangement.

Reported herein are mutual rearrangements in the whole series of seven bicapped-square antiprismatic closo-C2B8H10 by means of high-quality computations that disprove the earlier postulated dsd (diamond-square-diamond) scheme for these isomerizations. The experimentally existing closo-1,2-C2B8H10 was able to be converted to 1,6-, and 1,10-isomers by pyrolysis, and the dsd (diamond-square-diamond) mechanism was offered as an explanation of these processes. However, these computations disprove the postulated dsd scheme for these isomerizations that take place in the ten-vertex closo series. Experimentally observed thermal rearrangements, both in the parent and substituted closo-1,2-C2B8H10, closo-1-CB9H10-, and closo-B10H102-, indirectly support these refined computations. All these processes are based on the new concept of the so-called Z-mechanism, being consistent with a transition state of a boat shape with an open hexagonal belt that results from the initial breakage of three bonds. Such bond breakings and the consequent bond formations bring to mind the shape of the letter Z. In effect, the pattern of multicenter bonding shifts from reactant through a transition state to product. The molecular rearrangements that are available experimentally favour either the axial or equatorial isomers, and this ratio depends on temperature and the type of cluster and its substitution.

Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase.

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity.

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

Druggable Hot Spots in the Schistosomiasis Cathepsin B1 Target Identified by Functional and Binding Mode Analysis of Potent Vinyl Sulfone Inhibitors.

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

Benchmark Data Sets of Boron Cluster Dihydrogen Bonding for the Validation of Approximate Computational Methods.

The success of approximate computational methods, such as molecular mechanics, or dispersion-corrected density functional theory, in the description of non-covalent interactions relies on accurate parameterizations. Benchmark data sets are thus required. This area is well developed for organic molecules and biomolecules but practically non-existent for boron clusters, which have been gaining in importance in modern drug as well as material design. To fill this gap, we have introduced two data sets featuring the most common non-covalent interaction of boron clusters, the dihydrogen bond, and calculated reference interaction energies at the "golden standard" CCSD(T)/CBS level. The boron clusters studied interact with formamide, methanol, water and methane at various distances and in two geometrical arrangements. The performance of the tested approximate methods is variable and recommendations for further use are given.