Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

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

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 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 P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Brisbane Dental Hospital and Queensland University Dental College Building Alias The Palace: Heritage Listing.

The National Trust of Queensland placed the Brisbane Dental Hospital and Queensland College of Dentistry Building, alias The Palace, on the National Trust of Queensland Register in April 1997. This action generated no statutory consequences. Within days, the trust nominated The Palace for listing on the Queensland Heritage Register. Under the terms of the Queensland Heritage Act 1992, this nomination could have impeded an imminent $2-million redevelopment within The Palace. Two years later, the Queensland Heritage Council entered The Palace on the Queensland Heritage Register. This procedural delay was unusual and occurred in an era of post-Fitzgerald bureaucratic reform, federal cutbacks to funding for public dental services, tenuous political control of state government and widespread community support for heritage protection. The authors use historical methods to disclose and analyze hitherto inaccessible evidence relating to the delay in the listing. They argue that, against a backdrop of potential controversy, a small band of networked, organized and resolute administrators and Palace-based personnel, achieved the redevelopment. Astute tactics, concurrent rebuilding of health infrastructure, ministerial resolve, the nature of the act, public demand for dental services, the timing of the redevelopment and the political circumstances influenced the outcome.

Public Dental Services, Queensland, Australia: Charles Octavius Vidgen.

Charles Octavius Vidgen was the Superintendent of the Brisbane Dental Hospital, c1917-1945. Hitherto, commentators' reviews rely on imposing but narrow streams of evidence to either ignore Vidgen's influence on the dental profession or portray it as both peripheral and controversial. In this account, the authors use historical method to provide a revisionist account of Vidgen's professional profile and, to a lesser extent, a character resurrection. Vidgen was probably introverted. His orientation relating to dental education became obsolete, inappropriate and disruptive. Vidgen's actions, beliefs and values incurred sustained and organized opposition from academe, the Australian Dental Association Queensland Branch, the Odontological Society of Queensland and some private practitioners. The sociopolitical context, namely the Great Depression and affiliated reconstruction, the community's demand for government-administered dental services, World War II, twenty-five years of continuous Australian Labor Party government in Queensland, Edward Hanlon's authoritarianism and the emergence of a welfare state were also relevant to Vidgen's becoming a nonconformist, nonjoiner and an outcast. However, the authors posit that, for the socially disadvantaged and the regionally and remotely domiciled, Vidgen was a humanitarian and a quiet social reformer who, under Hanlon's authority and tutelage, pioneered enduring changes to the delivery of dental services across Queensland.

Public Dental Services, Queensland: Alan Thomas Robertson.

Alan Thomas Robertson's career as Assistant Superintendent Brisbane Dental Hospital [1927-1945] and Acting Superintendent [1945-1946] spanned difficult times. In Victoria, against a backdrop of family tragedy and World War I, Robertson achieved distinguished academic and war-service records. Following the move to Queensland, Robertson either experienced or witnessed the Great Depression, World War II and affiliated paradigm shifts in government policy, dental education and the system of the delivery of dental services. Within this context, the actions of Hanlon, Vidgen and Hoole overshadowed Robertson's brief but meaningful contribution to the Australian Dental Association Queensland Branch, his diligent nineteen years of service to the Brisbane Dental Hospital [BDH] and its patients, his pioneering of general anesthesia and his perennial commitment to undergraduate and continuing dental education. Robertson's career was neither financially lucrative nor acclaimed. Despite his overt patriotism, leadership potential, academic profile and experience, seniority and service, Robertson's appointment as Superintendent at the BDH was only an interim measure. A brief career in an entrepreneurial private practice ended in professional isolation followed by tragedy. The authors present a revisionist interpretation of Robertson's career. This narrative conveys messages for human resource managers in both academe and health departments.

Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Aß-Fibrinogen Interaction and Aß-Induced Contact System Activation.

Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer's disease (AD). Previous work has shown that the interaction between fibrinogen and ß-amyloid (Aß), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since Aß oligomers have a much stronger affinity for fibrinogen than Aß monomers, we tested whether amyloid aggregation inhibitors could block the Aß-fibrinogen interaction and found that some Aß aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy toward the Aß-fibrinogen interaction but also retained its potency toward the Aß42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated Aß42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the Aß-fibrinogen interaction and Aß aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as Aß aggregation-driven pathology in AD.

Structural and conformational determinants of macrocycle cell permeability.

Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.

Public Dental Services, Queensland: Alfred James Hoole.

Historians have given limited attention to the genesis and evolution of public dental services across Queensland. The Secretary [Minister] for Home Affairs and later Premier, Edward 'Ned' Hanlon, was the political architect of accessible public hospital and dental facilities. However it was administrator and dentist, Alfred James Hoole, who orchestrated the practical details in the field. Hoole developed an extensive and successful government-administered, hospital-based dental service that, in terms of reach and workforce, was the contemporaneous leader in Australia. These clinics and affiliated school dental services delivered treatment to a disproportionately high percentage of socially disadvantaged and remotely domiciled Queenslanders. Hoole's career progression from Superintendent of the Brisbane Dental Hospital to Director of Dental Services is remarkable for its achievements, consequences, competency and duration. It originated from a limited secondary education and traversed the bitter political split of 1957, changes of government, minister and fiscal policy, health adversity and opposition from private practitioners. Hoole, an anointed leader, a ministerial confidant and a pragmatist, served on authorities and institutions that shaped the future of dental education and dental practice across the state. Forty-five years after his death, Hoole's contribution to the administration of public dental services in Queensland remains unrivalled.

Public Dental Services, Queensland: Edward "Ned" Michael Hanlon.

Within the Australian context, commentators often portray the Queensland system of delivery of public dental services as state-specific. A poorly explored dimension within this narrative is the contribution from Ned Hanlon. The authors use historical methods to address this inadequacy in the literature. The implementation of Hanlon's vision of a statewide government-administered dental service required dentists and infrastructure; both implicated legislative and administrative changes to dental education, hospital organization and local authority. In this way, there was an inexorable link between the genesis and evolution of the public hospital and public dental systems. Hanlon's motive was initially humanitarian but later implicated pragmatism, state development and Queensland chauvinism. Hanlon's actions were autocratic, authoritarian and populist. He pursued regionalism, states rights and state development. The post-depression and post-war timing, together with the ubiquity of dental caries and the nature of the dental profession, facilitated Hanlon's success. A nascent and emerging dental profession was powerless, out of touch with public thinking and hindered by the legislative framework that controlled dentists' registration. The Hanlon-dentist encounters became an intersection of conflicting values; idealism and tradition versus pragmatism and innovation. Whatever the perceived inadequacies in Hanlon's methods, his contribution to public dentistry across Queensland remains remarkable.

The Brisbane Dental Hospital Building: "The Palace" An Era Ends.

Constitutional, educational, humanitarian and political considerations underpinned the design and construction of the Brisbane Dental Hospital Building, often colloquially referred to as "The Palace." The Queensland Heritage Council's listing of the Brisbane Dental Hospital Building on The Queensland Heritage Register in 1999 confirms the cultural significance of Nowland's architectural signature, the historical importance of the Wickham Park precinct and prior students' connection with the building. Influences on decisions determining the location, grand design and timing of construction of the Brisbane Dental Hospital Building emanated from a far bigger and largely unrecorded political picture. The authors argue that the political context in two tiers of government, the timing and nature of the proposal, town planning issues, the exigencies of the caries epidemic and Forgan Smith's post-Depression economic reconstruction across Queensland underpinned the project. Hanlon's personal attributes and disdain for the autonomy of the dental profession, together with his desire to reform dental education and to establish statewide government-administred dental clinics, were also relevant. Accordingly, the BDHD portrayed aspiration, purpose, symbolism, and vision. This paper, essentially an integration of dental and mainstream history, assembles and analyzes hitherto scattered and unpublished evidence to fill a gap in the current literature.

Synthesis of stereochemically and skeletally diverse fused ring systems from functionalized C-glycosides.

A diversity-oriented synthesis (DOS) strategy was developed for the synthesis of stereochemically diverse fused-ring systems containing a pyran moiety. Each scaffold contains an amine and methyl ester for further diversification via amine capping and amide coupling. Scaffold diversity was evaluated in comparison to previously prepared scaffolds by a shape-based principal moments of inertia (PMI) analysis.

Synthesis and profiling of a diverse collection of azetidine-based scaffolds for the development of CNS-focused lead-like libraries.

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

A Chemical Strategy toward Novel Brain-Penetrant EZH2 Inhibitors.

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors.

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

Novel Pure αVß3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVß3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVß3-mediated cell adhesion to αVß3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVß3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

Identification of Highly Specific Diversity-Oriented Synthesis-Derived Inhibitors of Clostridium difficile.

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.