Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia.

Shape-Based Virtual Screening of a Billion-Compound Library Identifies Mycobacterial Lipoamide Dehydrogenase Inhibitors.

Lpd (lipoamide dehydrogenase) in (Mtb) is required for virulence and is a genetically validated tuberculosis (TB) target. Numerous screens have been performed over the last decade, yet only two inhibitor series have been identified. Recent advances in large-scale virtual screening methods combined with make-on-demand compound libraries have shown the potential for finding novel hits.

Enhancing Hit Discovery in Virtual Screening through Absolute Protein-Ligand Binding Free-Energy Calculations.

In the hit identification stage of drug discovery, a diverse chemical space needs to be explored to identify initial hits. Contrary to empirical scoring functions, absolute protein-ligand binding free-energy perturbation (ABFEP) provides a theoretically more rigorous and accurate description of protein-ligand binding thermodynamics and could, in principle, greatly improve the hit rates in virtual screening. In this work, we describe an implementation of an accurate and reliable ABFEP method in FEP+.

Structure-Activity Relationship Studies of Antimalarial Proteasome Inhibitors─Part II.

With increasing reports of resistance to artemisinins and artemisinin-combination therapies, targeting the proteasome is a promising strategy for antimalarial development. We recently reported a highly selective proteasome inhibitor with anti-malarial activity in the humanized mouse model. To balance the permeability of the series of macrocycles with other drug-like properties, we conducted further structure-activity relationship studies on a biphenyl ether-tethered macrocyclic scaffold.

Induced-Fit Docking Enables Accurate Free Energy Perturbation Calculations in Homology Models.

Homology models have been used for virtual screening and to understand the binding mode of a known active compound; however, rarely have the models been shown to be of sufficient accuracy, comparable to crystal structures, to support free-energy perturbation (FEP) calculations. We demonstrate here that the use of an advanced induced-fit docking methodology reliably enables predictive FEP calculations on congeneric series across homology models ≥30% sequence identity. Furthermore, we show that retrospective FEP calculations on a congeneric series of drug-like ligands are sufficient to discriminate between predicted binding modes.

Design, Synthesis, and Optimization of Macrocyclic Peptides as Species-Selective Antimalaria Proteasome Inhibitors.

With over 200 million cases and close to half a million deaths each year, malaria is a threat to global health, particularly in developing countries. , the parasite that causes the most severe form of the disease, has developed resistance to all antimalarial drugs. Resistance to the first-line antimalarial artemisinin and to artemisinin combination therapies is widespread in Southeast Asia and is emerging in sub-Saharan Africa.

Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance.

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P.

In Vitro and In Vivo Inhibition of the Phosphopantetheinyl Transferase PptT by Amidinoureas.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in . The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring.

Fine Sampling of Sequence Space for Membrane Protein Structural Biology.

We describe an enhancement of traditional genomics-based approaches to improve the success of structure determination of membrane proteins. Following a broad screen of sequence space to identify initial expression-positive targets, we employ a second step to select orthologs with closely related sequences to these hits. We demonstrate that a greater percentage of these latter targets express well and are stable in detergent, increasing the likelihood of identifying candidates that will ultimately yield structural information.

Macrocyclic Peptides that Selectively Inhibit the Proteasome.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs.

Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the β5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P.

Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the proteasome (Pf20S) β5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against strains from patients in Africa. The β5 inhibitors synergize with a β2 inhibitor in vitro and in mice and with artemisinin.

Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c.

Identification of a Novel 1,2,3,4-Tetrahydrobenzo[b][1,6]naphthyridine Analogue as a Potent Phosphodiesterase 5 Inhibitor with Improved Aqueous Solubility for the Treatment of Alzheimer's Disease.

Phosphodiesterase 5 (PDE5) hydrolyzes cyclic guanosine monophosphate (cGMP) leading to increased levels of the cAMP response element binding protein (CREB), a transcriptional factor involved with learning and memory processes. We previously reported potent quinoline-based PDE5 inhibitors (PDE5Is) for the treatment of Alzheimer's disease (AD). However, the low aqueous solubility rendered them undesirable drug candidates.

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies.

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells.

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP.

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N-terminal "leader" sequence.

Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.

Structural and energetic determinants of adhesive binding specificity in type I cadherins.

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities.

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle.

The discovery of new cyanobactins from Cyanothece PCC 7425 defines a new signature for processing of patellamides.

Cyanobactins, including patellamides, are a group of cyanobacterial post-translationally modified ribosomal cyclic peptides. The final product should in theory be predictable from the sequence of the precursor peptide and the associated tailoring enzymes. Understanding the mechanism and recognition requirements of these enzymes will allow their rational engineering.

Nectin ectodomain structures reveal a canonical adhesive interface.

Nectins are immunoglobulin superfamily glycoproteins that mediate intercellular adhesion in many vertebrate tissues. Homophilic and heterophilic interactions between nectin family members help mediate tissue patterning. We determined the homophilic binding affinities and heterophilic specificities of all four nectins and the related protein nectin-like 5 (Necl-5) from human and mouse, revealing a range of homophilic interaction strengths and a defined heterophilic specificity pattern.

The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain.

Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically.

Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca²+-free interdomain linkers.

Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from Drosophila N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1') of the mature protein.

Transforming binding affinities from three dimensions to two with application to cadherin clustering.

Membrane-bound receptors often form large assemblies resulting from binding to soluble ligands, cell-surface molecules on other cells and extracellular matrix proteins. For example, the association of membrane proteins with proteins on different cells (trans-interactions) can drive the oligomerization of proteins on the same cell (cis-interactions). A central problem in understanding the molecular basis of such phenomena is that equilibrium constants are generally measured in three-dimensional solution and are thus difficult to relate to the two-dimensional environment of a membrane surface.