Crystal structure and ligandability of the 14-3-3/pyrin interface.

Overactivation of Pyrin is the cause of the inflammatory diseases Mediterranean Fever and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Binding of 14-3-3 proteins reduces the pro-inflammatory activity of Pyrin, hence small molecules that stabilize the Pyrin/14-3-3 complex could convey an anti-inflammatory effect. We have solved the atomic resolution crystal structures of phosphorylated peptides derived from PyrinpS208 and PyrinpS242 - the two principle 14-3-3 binding sites in Pyrin - in complex with 14-3-3 and analyzed the ligandability of these protein-peptide interfaces by crystal-based fragment soaking. The complex between 14-3-3 and PyrinpS242 appears to be much more amenable for small-molecule binding than that of 14-3-3/PyrinpS208. Consequently, only for the 14-3-3/PyrinpS242 complex could we find an interface-binding fragment, validating protein crystallography and fragment soaking as a method to evaluate the ligandability of protein surfaces.

Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences.

Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this 'oligonucleotide-linked topoisomerase inhibitor' (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIß. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery.

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

The discovery of potent and selective kynurenine 3-monooxygenase inhibitors for the treatment of acute pancreatitis.

A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

The effect of albumin leakage in hemodialysis patients on redox status of serum albumin.

Human mercaptoalbumin (HMA) is a reduced form of albumin that is associated with cardiovascular disease in dialysis patients. Albumin-leaky hemodialysis (HD) is increasingly recognized as a gold standard therapy because it is correlated with better prognosis compared to conventional HD. However, albumin-leaky HD induces low serum albumin concentration because of albumin leakage, which is a classical risk factor for mortality. The aim of this study was to explain the preferable prognosis in patients undergoing albumin-leaky HD with low serum albumin concentration. Ten HD patients were enrolled. They were preconditioned with albumin-non-leaky HD (mean albumin leakage: 1.0 g) for 2 months. Subsequently, albumin-leaky HD (9.1 g) was performed for 6 months, followed by relatively non-leaky HD (within 3.0 g). The ratio and level of HMA were evaluated. The amount of albumin leakage was related to the ratio of HMA, and inversely correlated with serum albumin concentration. The level of HMA was maintained regardless of albumin leakage. Regarding HMA level, a moderate amount of albumin leakage was acceptable. A stably maintained HMA level in albumin-leaky HD patients can contribute to preferable prognosis even if they have low serum albumin concentration.

Intracellular drug concentration and disposition--the missing link?

Improved understanding of the concentration of a potential drug molecule at the site of action in physiologically relevant cells or tissues has emerged as a key challenge in the early stages of drug discovery. Improved ability to carry out such studies with label-free methodology has the potential to improve understanding of drug uptake and trafficking and thus contribute to the reduction of rates of attrition in drug discovery.

Re-evaluation of Pre-pump Arterial Pressure to Avoid Inadequate Dialysis and Hemolysis: Importance of Prepump Arterial Pressure Monitoring in Hemodialysis Patients.

Prepump arterial pressure (PreAP) is monitored to avoid generating excessive negative pressure. The National Kidney Foundation K/DOQI clinical practice guidelines for vascular access recommend that PreAP should not fall below -250 mm Hg because excessive negative PreAP can lead to a decrease in the delivery of blood flow, inadequate dialysis, and hemolysis. Nonetheless, these recommendations are consistently disregarded in clinical practice and pressure sensors are often removed from the dialysis circuit. Thus far, delivered blood flow has been reported to decrease at values more negative than -150 mm Hg of PreAP. These values have been analyzed by an ultrasonic flowmeter and not directly measured. Furthermore, no known group has evaluated whether PreAP-induced hemolysis occurs at a particular threshold. Therefore, the aim of this study was to clarify the importance of PreAP in the prediction of inadequate dialysis and hemolysis. By using different diameter needles, human blood samples from healthy volunteers were circulated in a closed dialysis circuit. The relationship between PreAP and delivered blood flow or PreAP and hemolysis was investigated. We also investigated the optimal value for PreAP using several empirical monitoring methods, such as a pressure pillow. Our investigation indicated that PreAP is a critical factor in the determination of delivered blood flow and hemolysis, both of which occured at pressure values more negative than -150 mm Hg. With the exception of direct pressure monitoring, commonly used monitoring methods for PreAP were determined to be ineffective. We propose that the use of a vacuum monitor would permit regular measurement of PreAP.

Unlocking the secrets of the gatekeeper: methods for stabilizing and crystallizing GPCRs.

G-protein coupled receptors (GPCRs) are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the ß2 adrenergic receptor. Remarkably, at the time of writing, there have been an astonishing 18 further independent high-resolution GPCR structures published in the last five years (overall total of 68 structures in different conformations or bound to different ligands). Of particular note is the recent structure of the ß2 adrenergic receptor in complex with its cognate heterotrimeric G-protein revealing for the first time molecular details of the interaction between a GPCR and the complete G-protein. Together these structures have provided unprecedented detail into the mechanism of action of these incredibly important proteins. This review describes several key methodological advances that have made such extraordinarily fast progress possible.

Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest a dissociative mechanism that explains how ATP hydrolysis is coupled to domain motion.

DNA gyrase, a type II topoisomerase, regulates DNA topology by creating a double-stranded break in one DNA duplex and transporting another DNA duplex [T-DNA (transported DNA)] through this break. The ATPase domains dimerize, in the presence of ATP, to trap the T-DNA segment. Hydrolysis of only one of the two ATPs, and release of the resulting Pi, is rate-limiting in DNA strand passage. A long unresolved puzzle is how the non-hydrolysable ATP analogue AMP-PNP (adenosine 5'-[ß,γ-imido]triphosphate) can catalyse one round of DNA strand passage without Pi release. In the present paper we discuss two crystal structures of the Mycobacterium tuberculosis DNA gyrase ATPase domain: one complexed with AMP-PCP (adenosine 5'-[ß,γ-methylene]triphosphate) was unexpectedly monomeric, the other, an AMP-PNP complex, crystallized as a dimer. In the AMP-PNP structure, the unprotonated nitrogen (P-N=P imino) accepts hydrogen bonds from a well-ordered 'ATP lid', which is known to be required for dimerization. The equivalent CH2 group, in AMP-PCP, cannot accept hydrogen bonds, leaving the 'ATP lid' region disordered. Further analysis suggested that AMP-PNP can be converted from the imino (P-N=P) form into the imido form (P-NH-P) during the catalytic cycle. A main-chain NH is proposed to move to either protonate AMP-P-N=P to AMP-P-NH-P, or to protonate ATP to initiate ATP hydrolysis. This suggests a novel dissociative mechanism for ATP hydrolysis that could be applicable not only to GHKL phosphotransferases, but also to unrelated ATPases and GTPases such as Ras. On the basis of the domain orientation in our AMP-PCP structure we propose a mechanochemical scheme to explain how ATP hydrolysis is coupled to domain motion.

Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells.

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

Adapting the Cultural Formulation Interview for the Military.

OBJECTIVE: U.S. military service members, veterans, and their families increasingly seek care from providers with limited knowledge of military culture. The 16-item core DSM-5 Cultural Formulation Interview (CFI) was designed to integrate cultural factors into assessment and treatment of mental disorders. Although the CFI was designed for use with all patients, it is unknown whether the CFI adequately assesses military culture. The authors describe a methodology to determine the need for specific CFI versions and how to create a version for use with persons affiliated with the military. METHODS: Published articles on cultural competence in the military were systematically reviewed. Cultural domains were abstracted from each article, inductively coded, and hierarchically organized for assessment against the core CFI. A military CFI was created with additional implementation instructions, questions, and probes when the core CFI was inadequate for eliciting relevant cultural domains. RESULTS: Sixty-three articles were included. Coding revealed 22 military culture domains, of which only five would be elicited in the core CFI without additional guidance. Twelve of 16 questions in the core CFI required additional instructions, five benefited from question edits, and 10 needed additional probing questions. On the basis of these results, the authors crafted a military version of the CFI for service members, veterans, and their families. CONCLUSIONS: The military CFI for clinicians assesses aspects of military culture that are not comprehensively evaluated through the core CFI. The development process described in this article may inform the creation of other versions when the core CFI does not comprehensively assess cultural needs for specific populations.

Lessons in Transcellular Membrane Transport Re-Learned.

A brief note on the studies we have conducted on total and free drug concentration of thousands of drug discovery compounds in HeLa cells as measured by an approach inspired by the work of Professor Per Arturrsson. We conclude that the familiar QSAR equations of Corwin Hansch which were modelled as a bell shape by using logP and -logP2 terms can be similarly seen in our results and this can be interpreted with the aid of chromatographic Immobilised Artificial Membrane measurements. We also point out the differences between our measurements and those widely used based on Artificial Membrane Permeability Assays.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

The PROTACtable genome.

Proteolysis-targeting chimeras (PROTACs) are an emerging drug modality that may offer new opportunities to circumvent some of the limitations associated with traditional small-molecule therapeutics. By analogy with the concept of the 'druggable genome', the question arises as to which potential drug targets might PROTAC-mediated protein degradation be most applicable. Here, we present a systematic approach to the assessment of the PROTAC tractability (PROTACtability) of protein targets using a series of criteria based on data and information from a diverse range of relevant publicly available resources. Our approach could support decision-making on whether or not a particular target may be amenable to modulation using a PROTAC. Using our approach, we identified 1,067 proteins of the human proteome that have not yet been described in the literature as PROTAC targets that offer potential opportunities for future PROTAC-based efforts.

Early interventions in a US military FIRST episode psychosis program.

AIM: Naval Medical Center San Diego's Psychiatric Transition Program is a specialized first episode psychosis treatment program that delivers coordinated specialty care to military service members with psychotic disorders. Due to the unique military environment, military service members with first episode psychosis are hypothesized to receive care very early after the emergence of first psychotic symptoms, resulting in significantly reduced duration of untreated psychosis. This study's aim is to calculate the duration of untreated psychosis for patients enrolled in Naval Medical Center San Diego's Psychiatric Transition Program (NMCSD PTP) from 01JUL2014-31DEC2016. METHODS: Patients included in this study had a diagnosis of schizophreniform disorder (13.04%), schizophrenia (43.48%), schizoaffective disorder (8.70%), other specified schizophreniform disorder (30.43%), or brief psychotic disorder (4.35%) upon discharge from military service and NMCSD PTP. Duration of untreated psychosis was defined as the interval from emergence of positive psychotic symptoms to antipsychotic medication initiation. Duration of untreated psychosis was measured through retrospective review of the electronic medical record. A total of 69 subjects in the Naval Medical Center San Diego's Psychiatric Transition Program met inclusion criteria. Mean and median values as well as standard deviations were calculated for all included subjects. RESULTS: The mean duration to scheduled (non-PRN) neuroleptic medication was 37 days (median: 4 days). The mean duration to PRN neuroleptic medication was 21 days (median: 2 days). CONCLUSIONS: These data support our view that the structure of the military and military healthcare system markedly shortens the DUP for military service members who experience first episode psychosis.

Approaches to target tractability assessment - a practical perspective.

The assessment of the suitability of novel targets to intervention by different modalities, e.g. small molecules or antibodies, is increasingly seen as important in helping to select the most progressable targets at the outset of a drug discovery project. This perspective considers differing aspects of tractability and how it can be assessed using in silico and experimental approaches. We also share some of our experiences in using these approaches.

Progressive renal insufficiency related to ALK inhibitor, alectinib.

Alectinib is a second generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor and is generally effective and tolerated in patients who have demonstrated disease progression or adverse effects while on the first generation inhibitor, crizotinib. ALK inhibitors can cause a reversible chronic increase of serum creatinine concentration; however, they rarely induce progressive renal insufficiency. We herein report a case of a 68-year-old woman diagnosed with ALK-positive advanced non-small cell lung cancer and who received ALK inhibitors. Due to dysgeusia and transaminitis, her medication was switched from crizotinib to alectinib. Rapid progressive glomerulonephritis developed 1 year after the initiation of alectinib treatment. A renal biopsy revealed unique kidney lesions in both tubules and glomeruli. Glucocorticoid therapy partially reversed kidney impairment. However, re-administration of alectinib caused kidney dysfunction, which was improved by the cessation of alectinib. Our case suggests that much attention should be paid to kidney function when using ALK inhibitors.