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Recently reported inhibitors developed against targets from Mycobacterium absecessus (Mab).

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. We identify a biophysically, structurally and phenotypically validated lead compound,CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells,CAM2602synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a structural and chemical blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

Funder: Division of Intramural Research, National Institute of Allergy and Infectious Diseases; Id: http://dx.doi.org/10.13039/100006492, Funder: Cambridge Trust; Id: http://dx.doi.org/10.13039/501100003343, Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

The delivery of lipophilic dyes, such as BODIPY 505/515, to cells is often hindered by their low aqueous solubility, necessitating the use of organic solvents to facilitate the delivery, which unfortunately compromises the viability of the cells. In this work, we demonstrate the generation of novel composite hydrogel microparticles loaded with BODIPY 505/515, which can be used to deliver the dye to microalgal cells to stain the intracellular lipids. The microparticles were prepared by combining polymeric micelles with hydrogel technology to obtain microparticles of enhanced loading capacity. The generated hydrogel microparticles were tested by incubation with Phaeodactylum tricornutum algal cells. The cells were rapidly and successfully stained by the dye-containing microparticles, and their viability was not affected by the staining process. The method can also be used to stain other types of microalgal cells, such as Nannochloropsis gaditana cells. We therefore believe that this work offers a versatile and useful solution to important cell-staining problems in biotechnology.

Fragment-based drug discovery (FBDD) is a method of identifying small molecule hits that can be elaborated rationally through fragment growing, merging and linking, to afford high-affinity ligands for biological targets. Despite the promised theoretical potential of fragment linking, examples are still surprisingly sparse and remain overshadowed by the successes of fragment growing. The aim of this review was to outline a number of key examples of fragment-linking strategies and discuss their strengths and limitations. Structure-based approaches including X-ray crystallography and in silico methods of fragment optimization are discussed, as well as fragment linking guided by NMR experiments. Target-guided approaches, exploiting the biological target to assemble its own inhibitors through dynamic combinatorial chemistry (DCC) and kinetic target-guided synthesis (KTGS), are identified as alternative efficient methods for fragment linking.

16 p.-8 fig.-4 tab., The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes., This work was funded by Bill and Melinda Gates Foundation HIT-TB (OPP1024021) and SHORTEN-TB (OPP1158806). PG was funded by a Gates Cambridge Scholarship. TLB is funded by the Wellcome Trust (Wellcome Trust Investigator Award 200814_Z_16_Z: RG83114). ST acknowledges SC1GM140968 grant from National Institute of Health to support this work.

Mycobacterium abscessus (Mab) is a rapidly growing multidrug-resistant species among nontuberculous mycobacteria (NTM). Pulmonary infections caused by M. abscessus are difficult to treat and often result in an accelerated condition and premature death of immunosuppressed patients such as those with cystic fibrosis, putting them at a greater risk of other infections and increasing the urgency of developing a novel class of antibiotics. Here, we explore the use of toxin and antitoxin (TA) as an interesting and promising new class of target in a fragment-based drug-discovery approach. A de novo structure of Mab3862, an antitoxin of type 2 TA class in M. abscessus was elucidated and followed by drug discovery work. Very small molecules (fragments) are used to bind the antitoxin and then elaborated into drug-sized molecule that potentially trigger conformational change to prevent formation of toxin-antitoxin complex. Biophysical screening methods and binding-mode guidance from in silico docking were conducted. Grouping of fragments based on the binding site, creating a pharmacophore model, can facilitate further studies for rational design of inhibitors. Although targeting the TA complex for developing antibiotics is relatively novel and challenging, this could possibly open the gate for exploring it as potential drug target. Targeting one of the TA pairs might not be completely bactericidal. However, using this approach strategically with other antibiotics for synergic effect might be effective for patients with a persistence phenotype requiring prolonged therapy.

Made available in DSpace on 2020-12-10T20:10:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-14 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Bill & Melinda Gates Foundation Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) RSC grant DAAD Cambridge Trust Emmanuel College Support Program for Foreign Ph.D. Student and Ibero-American Graduate Association EPSRC Ph.D. fellowship Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors. Univ Sao Paulo, Dept Microbiol, Inst Biomed Sci, BR-05508000 Sao Paulo, SP, Brazil Univ Estadual Campinas, Inst Biol, BR-13083862 Campinas, SP, Brazil Univ Cambridge, Dept Chem, Cambridge CB2 1EW, England State Univ Sao Paulo, IBILCE, Dept Biol, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Univ Valle, Dept Microbiol, Cali 760043, Colombia Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England Univ Warwick, Dept Chem, Coventry CV4 7AL, W Midlands, England Natl Lab Biosci, BR-13083100 Campinas, SP, Brazil Univ Sao Paulo, Fac Pharmaceut Sci, Dept Pharm, BR-05508000 Sao Paulo, SP, Brazil State Univ Sao Paulo, IBILCE, Dept Biol, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2010/15971-3 FAPESP: 2014/09188-8 FAPESP: 2018/00351-1 FAPESP: 13/15906-5 FAPESP: 17/25733-1 Bill & Melinda Gates Foundation: RG48788 MAAG/555 Bill & Melinda Gates Foundation: PHZF/121 CNPq: 442021/2014-3 RSC grant: RF-17-9399 Support Program for Foreign Ph.D. Student and Ibero-American Graduate Association: PAEDEx/AUIP2014

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Fiber-shaped soft constructs are indispensable building blocks for various 3D functional objects such as hierarchical structures within the human body. The design and fabrication of such hierarchically structured soft materials, however, are often challenged by the trade-offs between stiffness, toughness, and continuous production. Here, we describe a microfluidic platform to continuously fabricate double network hydrogel microfibers with tunable structural, chemical, and mechanical features. Construction of the double network microfibers is accomplished through the incorporation of dynamic cucurbit[n]uril host–guest interactions, as energy dissipation moieties, within an agar-based brittle network. These microfibers exhibit an increase in fracture stress, stretchability, and toughness by 2–3 orders of magnitude compared to the pristine agar network, while simultaneously gaining recoverable hysteretic energy dissipation without sacrificing mechanical strength. This strategy of integrating a wide range of dynamic interactions with the breadth of natural resources could be used in the preparation of functional hydrogels, providing a versatile approach toward the continuous fabrication of soft materials with programmable functions.

The ability to construct core-shell microcapsules has the potential to shift the paradigm in the development of new delivery systems for nutrients, cosmetics, and drugs. In this work, we demonstrate an application of focused surface acoustic wave (FSAW) microfluidics to produce microcapsules with a core-shell structure using one or two focused interdigital transducers (FIDTs) on the microfluidic device. Solid particles or liquid microdroplets without any special modification in multiphase laminar flow are driven by the acoustic radiation force arising from the FSAW, and cross the oil/water interface back and forth, which is not only suitable for generation of core-shell microcapsules with solid cores but also used for coating an aqueous microdroplet core with an oil shell. On this basis, more FIDTs can be added to the device to manufacture more layers of microcapsules if needed. Single-layer, two-layer, or even multi-layer microcapsules can be selectively fabricated. This work provides a promising and robust platform to construct core-shell microcapsules via FSAW microfluidics, which are suitable for a wide range of applications.

Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 μM (LE 0.35).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved lead-to-clinical candidate medicinal chemistry campaign.

We demonstrate a novel microdroplets generation method by using focused surface acoustic waves (FSAW). The method differs from previous work in the mechanism and the geometry structure of the chip, which is depend on FSAW rather than flow shearing. The acoustic radiation force arising from FSAW acts on the oil-water interface, breaking up the water into microdroplets whose size can be controlled by tuning the driving voltage and frequency of FSAW. This approach overcomes the limitation of microchannel structure and capillary number of the traditional microfluidic droplets formation methods, which enables a fully electrical control of microdroplets size, with a good uniformity. Further, the FSAW-induced microdroplets are used as templates to prepare microgels with uniform size, offering a new platform for the design and synthesis of monodispersed functional microspheres.

Herein we describe a colour encoding system based on sequentially-encoded colloidal crystal microdots within hydrogel microfibres. This approach exploits a microfluidic spinning technology to generate microfibres in which colloidal crystals, of tuneable size, interval distance, number and sequential array, are orthogonally and simultaneously embedded. The hydrogel microfibre is comprised of calcium alginate, which can gel rapidly, is transparent and soft, and serves as a scaffold for the colloidal crystals. Further, the structural colour of the colloidal crystal microdots is visible and spectroscopically readable, offering a reliable and versatile colour-coding toolbox. We demonstrate the scalability and tuneability of the microfluidic spinning approach for generating hydrogel microfibres that exhibit encoding units from single dot to multiple coloured dots. This paper provides the proof of concept study of developing the application of the colloidal crystals to construct a novel encoding system.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4′-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4′-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential of more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

Summary BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy., Graphical abstract, Highlights • CAM833 inhibits the protein-protein interaction between RAD51 and BRCA2 • Crystal structure shows CAM833 binds RAD51 at the same site as the BRCA2 FxxA motif • CAM833 blocks formation of RAD51 foci and filaments, preventing DNA repair • CAM833 potentiates cytotoxicity by IR and synergises with PARP1 inhibitors, Scott et al. describe CAM833, an inhibitor of DNA recombinase RAD51, that binds at the site normally occupied by the BRC repeats of the BRCA2 protein. Through blocking this protein-protein interaction, CAM833 is able to prevent RAD51-mediated homologous recombination and thus potentiate cancer cells to damage by radiation or PARP1 inhibition.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB., The bifunctional enzyme CoaBC catalyses the second and third step in the Coenzyme A (CoA) biosynthesis pathway and is of interest as a M. tuberculosis drug target. Here, the authors present the full-length crystal structure of Mycobacterium smegmatis CoaBC, which is regulated by CoA and CoA thioesters and forms a dodecamer and by performing a high-throughput screen they identify selective inhibitors of M. tuberculosis CoaB that bind to an allosteric site within CoaB.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.Abstract Figure

The ability to construct core-shell microcapsules has the potential to shift the paradigm in the development of new delivery systems for nutrients, cosmetics, and drugs. In this work, we demonstrate an application of focused surface acoustic wave (FSAW) microfluidics to produce microcapsules with a core-shell structure using one or two focused interdigital transducers (FIDTs) on the microfluidic device. Solid particles or liquid microdroplets without any special modification in multiphase laminar flow are driven by the acoustic radiation force arising from the FSAW, and cross the oil/water interface back and forth, which is not only suitable for generation of core-shell microcapsules with solid cores but also used for coating an aqueous microdroplet core with an oil shell. On this basis, more FIDTs can be added to the device to manufacture more layers of microcapsules if needed. Single-layer, two-layer, or even multi-layer microcapsules can be selectively fabricated. This work provides a promising and robust platform to construct core-shell microcapsules via FSAW microfluidics, which are suitable for a wide range of applications.

SUMMARYBRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of

Tuberculosis (TB) remains a leading cause of mortality amongst infectious diseases worldwide. InhA, an enoyl ACP-reductase, has been the focus of numerous drug discovery efforts as this is the target of the first line pro-drug isoniazid. However, with resistance to this drug becoming more common the aim has been to find new clinical candidates that directly inhibit this enzyme and that do not require activation by the catalase peroxidase KatG, thus circumventing the majority of the resistance mechanisms. In this work, the screening and validation of a fragment library is described and development of the fragment hits using a fragment growing strategy was employed which led to the development InhA inhibitors with affinities of up to 250 nM.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, none have been developed to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that differs from other DHFR antifolates, thus opening perspectives for the development of novel and relevant MtDHFR inhibitors.

The recent advances in the supramolecular polymerization of synthetic building blocks in aqueous conditions has given rise to new artificial and biocompatible functional materials. However, despite the importance of spatially resolved self‐assembly for natural and artificial molecular machines, the spatial control of supramolecular polymerization with synthetic monomers has not been experimentally established yet. Here we describe a microfluidic‐regulated tandem process of supramolecular polymerization and droplet encapsulation to control the position of self‐assembled microfibrillar bundles of cyclic peptide nanotubes in water droplets. This method allowed the precise preferential localization of the fibres either at the interface or into the core of the droplets. UV absorbance, circular dichroism and fluorescence microscopy indicated that the microfluidic control of the stimuli (changes in pH or ionic strength) can be employed to adjust the packing degree and the spatial position of microfibrillar bundles of cyclic peptide nanotubes. Additionally, this spatially organized supramolecular polymerization of peptide nanotubes was applied in the assembly of highly ordered two‐dimensional droplet networks This work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R, CTQ2016-78423-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248). J. M. holds a Ramón y Cajal (RYC-2013-13784), an ERC-Stg (DYNAP-677786) and a Young Investigator Grant from the HFSP (RGY0066/2017). J. R. G. thanks to the mobility program (PRX17/00147) SI

Although materials with structural colours are known to be resistant to photobleaching, the damage caused by structural failure and lack of suitable fabrication methods limit their practical uses. Herein, confinement-mediated self-assembly of colloidal crystals in dynamic borate ester-based networks is demonstrated to produce spherical opal composites using a microfluidic device. The resulting optical beads are flowable and injectable, facilitating injection moulding of three-dimensionally well-defined macroscopic opals. Further, the opal composites formed in semi-spherical sessile microdroplets can serve as individual pixels for two-dimensional patterns integrating angle-independent structural colours and self-healing characteristics. These findings provide novel insights for the development of multi-scale opal constructs with ability to resist the physical damage and to increase the lifetime.

Tuberculosis (TB) remains a leading cause of mortality amongst infectious diseases worldwide. InhA, the target of the first line pro-drug isoniazid, an enoyl ACP-reductase, has been the focus of numerous drug discovery efforts attempting to find new clinical candidates that directly inhibit this enzyme and that do not require activation by the catalase peroxidase KatG, thus circumventing the majority of the resistance mechanisms. In this work, we describe the screening and validation cascade of a fragment library and employ a fragment growing strategy using structure-guided fragment-based design to develop potent InhA inhibitors.

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH.

With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a nonconserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations.

With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in