Optimization of lead compounds into on-demand, nonhormonal contraceptives: leveraging a public–private drug discovery institute collaboration†

Abstract Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.

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Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

10.1101/2021.04.27.441671 ◽

2021 ◽

Author(s):

Melanie Balbach ◽

Lubna Ghanem ◽

Thomas Rossetti ◽

Navpreet Kaur ◽

Carla Ritagliati ◽

...

Keyword(s):

Adenylyl Cyclase ◽

Acrosome Reaction ◽

Human Sperm ◽

Comprehensive Analysis ◽

Multiple Points ◽

Soluble Adenylyl Cyclase ◽

Mouse Sperm ◽

On Demand ◽

Sperm Functions

AbstractSoluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperm’s journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

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Novel Small-Molecule Inhibitors of Arylamine N-Acetyltransferases: Drug Discovery by High Throughput Screening

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/138620711794474051 ◽

2011 ◽

Vol 14 (2) ◽

pp. 117-124 ◽

Cited By ~ 17

Author(s):

Isaac M. Westwood ◽

Akane Kawamura ◽

Angela J. Russell ◽

James Sandy ◽

Stephen G. Davies ◽

...

Keyword(s):

Drug Discovery ◽

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitors

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A Central Role for Biophysics in Cancer Drug Discovery - Development of Candidate Small Molecule Inhibitors in Mutant KRas

Biophysical Journal ◽

10.1016/j.bpj.2017.11.214 ◽

2018 ◽

Vol 114 (3) ◽

pp. 30a-31a ◽

Cited By ~ 1

Author(s):

Andrea Gohlke ◽

Justin Bower ◽

Peter N. Brown ◽

Ken S. Cameron ◽

Martin Drysdale ◽

...

Keyword(s):

Drug Discovery ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Cancer Drug ◽

Cancer Drug Discovery

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Development of Small Molecule Inhibitors of the AML1-ETO and CBFβ-SMMHC Oncoproteins.

Blood ◽

10.1182/blood.v110.11.1591.1591 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1591-1591

Author(s):

Jolanta E. Grembecka ◽

Kristin Graf ◽

Yali Kong ◽

Michael Douvas ◽

Tomasz Cierpicki ◽

...

Keyword(s):

Fusion Protein ◽

Cell Line ◽

Small Molecule ◽

Protein Interaction ◽

Small Molecule Inhibitors ◽

Leukemia Cell Line ◽

Runt Domain ◽

Protein Protein Interaction ◽

Lead Compounds ◽

Binding Interface

Abstract Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFα) and CBFβ subunits which are essential for normal blood cell development. CBFβ functions to increase the DNA-binding of the RUNX1 subunit 20–40 fold and to protect the RUNX1 subunit against ubiqitination and proteasome degradation, making this protein-protein interaction critical for CBF function. Two of the most common translocations involving the subunits of CBF are the inv(16) and the t(8;21) which produce the chimeric proteins CBFβ-SMMHC and AML1-ETO, respectively, which are associated with the development of Acute Myeloid Leukemia (AML). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFβ activity in vivo and causes a blockage in normal hematopoiesis, predisposing for the development of leukemia. The interaction between CBFβ and AML1-ETO is critical for its function, therefore treatments targeting AML1-ETO and blocking its interaction with CBFβ are highly likely to be therapeutically beneficial. The CBFβ-SMMHC fusion protein causes dysregulation of CBF function by means of anomalously tight binding to RUNX1. Since binding to RUNX1 is required for the dysfunction associated with CBFβ-SMMHC, this interaction represents an excellent target for inhibition as a potential therapeutic strategy. We have initiated efforts to develop small molecule inhibitors of the RUNX1-CBFβ interaction as possible therapeutics for the treatment of the associated leukemias. Both virtual screening searches, focused on the X-ray structures of RUNX1 Runt domain and CBFβ, and high-throughput screening of NCI (National Cancer Institute) and Maybridge fragment libraries were used to identify initial lead compounds interacting with these proteins and blocking heterodimerization of CBF. Compounds were tested experimentally by FRET (Fluorescence Resonance Energy Transfer) and ELISA for their inhibition of RUNX1-CBFβ interaction. This resulted in a number of initial lead compounds targeting either the Runt domain or CBFβ and inhibiting this protein-protein interaction. Based on the docking mode selected lead compounds were further optimized using medicinal chemistry approaches to increase their affinity and determine the structure-activity relationships (SAR). This resulted in several compounds with low micromolar affinity (IC50 < 10 μM) which effectively block the heterodimerization of CBF in vitro and in a cell-based assay. Interestingly, compounds targeting CBFβ bind to a site displaced from the binding interface for RUNX1 as shown by the NMR-based docking, i.e. these compounds function as allosteric inhibitors of this protein-protein interaction. The most potent compounds were tested either in the Kasumi-1 leukemia cell line harboring t(8;21) translocation or in the ME-1 cell line with inv(16), resulting in a blockage of proliferation, induction of apoptosis and differentiation of these cells. These compounds represent the first small molecule inhibitors targeting CBF and inhibiting this interaction. They represent good starting points for the development of therapeutically useful inhibitors. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO or CBFβ-SMMHC oncoproteins versus wild type proteins.

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Computational Improvement of Small-Molecule Inhibitors of Bacillus anthracis Protective Antigen Activation through Isostere-Based Substitutions

10.20944/preprints201912.0247.v1 ◽

2019 ◽

Author(s):

Sandra V. R. L. Silva ◽

Pedro J. Silva

Keyword(s):

Bacillus Anthracis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Protective Antigen ◽

Binding Modes ◽

Lead Compounds ◽

Computational Work ◽

Binding Cavity ◽

The Stability ◽

Dynamics Simulations

There has recently been interest in the development of small-molecule inhibitors of the oligomerization of Bacillus anthracis protective antigen for therapeutic use. Some of the proposed lead compounds have, however, unfavorable solubility in aqueous medium, which prevents their clinical use. In this computational work, we have designed several hundreds of derivatives with progressively higher hydrosolubility and tested their ability to dock the relevant binding cavity. The highest-ranking docking hits were then subjected to 125 ns-long simulations to ascertain the stability of the binding modes. Several of the potential candidates performed quite disappointingly , but two molecules showed very stable binding modes throughout the complete simulations. Besides the identification of these two promising leads, these molecular dynamics simulations allowed the discovery of several insights that shall prove useful in the further improvement of these candidate towards higher potency and stability.

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Nanomolar Inhibitors ofTrypanosoma bruceiRNA Triphosphatase

mBio ◽

10.1128/mbio.00058-16 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 7

Author(s):

Paul Smith ◽

C. Kiong Ho ◽

Yuko Takagi ◽

Hakim Djaballah ◽

Stewart Shuman

Keyword(s):

Drug Discovery ◽

Trypanosoma Brucei ◽

Small Molecule ◽

Active Sites ◽

Small Molecule Inhibitors ◽

Chemical Space ◽

Antiviral Drug ◽

Mrna Capping ◽

Rna Triphosphatase ◽

Triphosphate Tunnel Metalloenzyme

ABSTRACTEukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show thatTrypanosoma bruceiRTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.IMPORTANCEThe stark differences between the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 fromTrypanosoma brucei, is essential for growth of the parasite in culture and identifies, viain vitroscreening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.

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Identifying small-molecule lead compounds: The screening approach to drug discovery

Trends in Biotechnology ◽

10.1016/s0167-7799(00)88916-7 ◽

1995 ◽

Vol 13 (3) ◽

pp. 115-121 ◽

Cited By ~ 56

Author(s):

Paul Bevan ◽

Hamish Ryder ◽

Ian Shaw

Keyword(s):

Drug Discovery ◽

Small Molecule ◽

Lead Compounds ◽

Screening Approach

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Abstract LB-228: Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery

10.1158/1538-7445.am2017-lb-228 ◽

2017 ◽

Author(s):

Daniel Croft ◽

Stuart Francis ◽

Justin Bower ◽

Andrea Gohlke ◽

Gillian Goodwin ◽

...

Keyword(s):

Drug Discovery ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Fragment Based Drug Discovery

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Niche-Based Screening Identifies Novel Small Molecules That Overcome Stromal Effects in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.571.571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 571-571

Author(s):

Shrikanta Chattopadhyay ◽

Alison L. Stewart ◽

Siddhartha Mukherjee ◽

Cherrie Huang ◽

Kimberly A. Hartwell ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Discovery ◽

Small Molecules ◽

Cell Line ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Small Molecule Inhibitors ◽

Novel Therapeutic

Abstract Abstract 571 Despite advances in the treatment of multiple myeloma (MM), this disease remains incurable and novel therapeutic strategies are urgently needed. Ideal strategies would overcome resistance factors from the bone-marrow microenvironment (niche) since a variety of inhibitors are rendered less effective by bone-marrow stromal cells (BMSCs) of the MM niche (McMillin et al., Nat Med. 2010 Apr;16(4):483–9). Drug discovery often entails a target-based approach but identifying targets in MM is challenging because of its complex genome and multiple niche interactions. We used a chemical biology approach in which small-molecule inhibitors of MM cells, grown within their niche, are first identified and then used to discover targets within MM or its niche. These compounds also serve as leads for future drug discovery. To model myeloma/niche interactions, we chose an MM cell line MOLP5 that has an obligate dependence on BMSCs to maintain viability. Small-molecule inhibitors were identified by screening ∼25,000 structurally diverse small molecules on GFP-labeled MOLP5 cells co-cultured with primary BMSCs derived from hip replacement samples. MOLP5 growth inhibition was measured by quantifying GFP(+) cells with automated high-throughput microscopy. About 800 hits were counter-screened on BMSCs alone to exclude non-specifically toxic compounds. The remaining 182 MOLP5-selective inhibitors were then tested on 2 other GFP-labeled MM cell-lines, MM1S and INA6, in the presence or absence of BMSCs to exclude compounds that are less effective in the presence of BMSCs. The 64 compounds that overcome BMSC resistance were tested on CD34+ human hematopoietic progenitors to prioritize compounds with selectivity between MM and normal blood cells. The 8 compounds that met these criteria fell into 3 categories: 1) compounds with equal activity in the presence or absence of BMSCs (overcome stromal resistance); 2) compounds with selectivity for BMSC-dependent MOLP5 cells (block stromal viability factors); and 3) compounds with increased activity in the presence of BMSCs (enhance stromal inhibitory factors). Because most efficacious clinical compounds like bortezomib act like compounds in category 1, compound BRD9876 was chosen from this category for mechanistic studies. Gene-expression profiling of BRD9876-treated MM1S cells suggested possible links to mitotic arrest and cell cycle analyses revealed a rapid accumulation of cells in the G2/M phase. Treated cells were stained for the mitotic spindle protein α-tubulin and found to exhibit an aberrant mono-astral mitotic phenotype, reminiscent of the kinesin-5 (Eg5; KIF11) inhibitor monastrol. This was encouraging because a kinesin-5 inhibitor ARRY-520 has shown promising durable responses in multiple myeloma (Shah et al, ASH Annual Meeting 2011; Abstract 1860). To determine if BRD9876 was a kinesin-5 inhibitor, a BRD9876-resistant sub-line of MM1S was developed and the kinesin-5 gene sequenced. BRD9876-resistant cells have a novel kinesin-5 mutation (Y104C) at a site that is distant from the monastrol-binding pocket. Most kinesin-5 inhibitors in clinical development bind the monastrol pocket, and the BRD9876-resistant cells were not cross-resistant to one such inhibitor, ispinesib, suggesting a distinct mode of kinesin-5 inhibition by BRD9876. To identify biomarkers of sensitivity to BRD9876, quantitative dose/response measurements in 98 genetically characterized cell lines (Schreiber & co-workers, submitted) comprising a subset of the Cancer Cell Line Encyclopedia (CCLE) were analyzed. Unbiased analyses correlating genetic features with sensitivity revealed that mutations in the mitotic regulator WEE1 were associated with sensitivity to BRD9876. Validation studies comparing WEE1 mutant to wild-type cell lines confirmed enhanced sensitivity of mutant cells to both BRD9876 and ispinesib suggesting that WEE1 mutations could be a useful biomarker for different kinesin-5 inhibitors. In contrast, co-treatment of WEE1 WT cells with sub-toxic concentrations of the WEE1 inhibitor MK1775 led to marked enhancement of BRD9876 activity but had little effect on ispinesib activity, suggesting a unique synergistic relationship between WEE1 inhibitors and BRD9876. In summary, niche-based screening in multiple myeloma has revealed a novel therapeutic candidate and can complement other drug-discovery approaches against this disease. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

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